Polypeptides having lipase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having lipase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/614,508, filed Sep. 30, 2004, U.S. Provisional Application No.60/621,304, filed Oct. 21, 2004, and U.S. Provisional Application No.60/621,222, filed Oct. 21, 2004, which applications are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides having lipaseactivity and isolated polynucleotides encoding the polypeptides. Theinvention also relates to nucleic acid constructs, vectors, and hostcells comprising the polynucleotides as well as methods for producingand using the polypeptides.

2. Description of the Related Art

Triacylglycerol hydrolyzing enzymes are enzymes that catalyze thehydrolysis or formation of triglycerides. Triacylglycerol hydrolyzingenzymes are a versatile group of enzymes and often have more than oneactivity such as lipase, phospholipase, lysophospholipase, cholesterolesterase, cutinase, amidase, galactolipase, and other esterase type ofactivities. Which activity is the predominant activity will depend onthe application of the enzyme and the conditions.

Triacylglycerol hydrolyzing enzymes belong to the IUBMB EnzymeNomenclature 3.1.1. EC 3 refers to hydrolases, EC 3.1 refers to actingon ester bonds, and EC 3.1.1 refers to carboxylic ester hydrolases.Related enzymes are classified in EC 3.1.4, which refers to phosphoricdiester hydrolases.

Lipases (EC 3.1.1.3) are enzymes that catalyze the hydrolysis of a widerange of carboxy esters, e.g., triglycerides to release fatty acid.Esterases (EC 3.1.1.1) are enzymes that catalyze the hydrolysis ofwater-soluble carboxylic esters, including short-chain fatty acidtriglycerides, to produce an alcohol and a carboxylic acid anion.

Some lipases also have phospholipase activity and/or galactolipaseactivity (see, for example, U.S. Pat. No. 6,103,505 and U.S. Pat. No.6,852,346).

Phospholipases are enzymes that catalyze the hydrolysis of phospholipidswhich consist of a glycerol backbone with two fatty acids in the sn1 andsn2 positions, which is esterified with a phosphoric acid in the sn3position. The phosphoric acid may, in turn, be esterified to an aminoalcohol.

There are several types of phospholipases which catalyze the hydrolysisof the fatty acyl moieties. These phospholipases include phospholipaseA₁ (EC 3.1.1.32), phospholipase A₂ (EC 3.1.1.4), and lysophospholipase(EC 3.1.1.5). Phospholipase C (EC 3.1.4.3) and phospholipase D (EC3.1.4.4) hydrolyze the phosphoric acid group from a phospholipid, but donot hydrolyze fatty acids like phospholipase A₁, phospholipase A₂ andphospholipase B.

Phospholipase A₁ (EC 3.1.1.32) catalyzes the deacylation of one fattyacyl group in the sn1 position from a diacylglycerophospholipid toproduce lysophospholipid and fatty acid. Phospholipase A₂ (EC 3.1.1.4)catalyzes the deacylation of one fatty acyl group in the sn2 positionfrom a diacylglycerophospholipid to produce lysophospholipid and fattyacid. Lysophospholipase (EC 3.1.1.5), also known as phospholipase B,catalyzes the hydrolysis of the fatty acyl group in a lysophospholipid.Phospholipase C (EC 3.1.4.3) catalyzes the hydrolysis ofphosphatidylcholine to 1,2-diacylglycerol and choline phosphate.Phospholipase D (EC 3.1.4.4) catalyzes the hydrolysis of the terminalphosphate diester bond of phosphatidylcholine to produce choline andphosphatidic acid.

Galactolipases (EC 3.1.1.26) catalyze the hydrolysis of galactolipids byremoving one or two fatty acids.

Detergents formulated with lipolytic enzymes are known to have improvedproperties for removing fatty stains. For example, LIPOLASE™ (NovozymesA/S, Bagsværd, Denmark), a microbial lipase obtained from the fungusThermomyces lanuginosus (also called Humicola lanuginosa), has beenintroduced into many commercial brands of detergent. Lipases have alsobeen used in degumming processes and baking.

El-Shahed et al., 1988, Eqypt. J. Microbiol. 23: 357-372 and Mohawed etal., 1988, Eqypt. J. Microbiol. 23: 537-547 disclose two Aspergillusfumigatus lipases.

WO 03/12071 discloses a gene encoding a lipase from Aspergillusfumigatus.

Mayordomo et al., 2000, J. Agric. Chem. 48: 105-109 disclose theisolation, purification, and characterization of a cold-active lipasefrom Aspergillus nidulans.

Lipases have many commercial uses but very few lipases that work underapplication conditions and can be produced with high yields by microbialfermentation have been identified. There is a need in the art foralternative lipases with improved properties.

It is an object of the present invention to provide polypeptides havinglipase activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having lipaseactivity selected from the group consisting of:

(a) a polypeptide having an amino acid sequence which has at least 60%identity with the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, orSEQ ID NO: 16;

(b) a polypeptide which is encoded by a nucleotide sequence whichhybridizes under at least medium stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQID NO: 15, (ii) the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or(iii) a complementary strand of (i) or (ii); and

(c) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

The present invention also relates to isolated polynucleotides encodingpolypeptides having lipase activity, selected from the group consistingof:

(a) a polynucleotide encoding a polypeptide having an amino acidsequence which has at least 60% identity with the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16;

(b) a polynucleotide having at least 60% identity with the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:15; and

(c) a polynucleotide which hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii) the cDNA sequence contained inthe mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,or SEQ ID NO: 15, or (iii) a complementary strand of (i) or (ii).

In a preferred aspect, the mature polypeptide is amino acids 25 to 396of SEQ ID NO: 2, amino acids 25 to 283 of SEQ ID NO: 4, amino acids 20to 318 of SEQ ID NO: 6, amino acids 19 to 348 of SEQ ID NO: 8, aminoacids 25 to 393 of SEQ ID NO: 10, amino acids 20 to 294 of SEQ ID NO:12, amino acids 25 to 308 of SEQ ID NO: 14, or amino acids 26 to 404 ofSEQ ID NO: 16. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 73 to 1256 of SEQ ID NO: 1, nucleotides73 to 944 of SEQ ID NO: 3, nucleotides 58 to 1085 of SEQ ID NO: 5,nucleotides 55 to 1044 of SEQ ID NO: 7, nucleotides 73 to 1179 of SEQ IDNO: 9, nucleotides 58 to 1038 of SEQ ID NO: 11, nucleotides 73 to 1119of SEQ ID NO: 13, or nucleotides 76 to 1280 of SEQ ID NO: 15.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides.

The present invention also relates to methods for producing such apolypeptide having lipase activity comprising: (a) cultivating arecombinant host cell comprising a nucleic acid construct comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of using the polypeptideshaving lipase activity in detergents, degumming, and baking.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of nucleotides 1 to 72 of SEQ ID NO: 1, nucleotides 1 to 72of SEQ ID NO: 3, nucleotides 1 to 57 of SEQ ID NO: 5, nucleotides 1 to54 of SEQ ID NO: 7, nucleotides 1 to 72 of SEQ ID NO: 9, nucleotides 1to 57 of SEQ ID NO: 11, nucleotides 1 to 72 of SEQ ID NO: 13, ornucleotides 1 to 75 of SEQ ID NO: 15, wherein the gene is foreign to thenucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pSMO224.

FIG. 2 shows the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus fumigatus lipase (Aspergillus fumigatus lip1,SEQ ID NOs: 1 and 2, respectively).

FIG. 3 shows a restriction map of pAlLo1.

FIG. 4 shows a restriction map of pBM121b.

FIG. 5 shows a restriction map of pBM120a.

FIG. 6 shows a restriction map of pSMO218.

FIG. 7 shows a restriction map of pSMO223.

FIG. 8 shows the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus fumigatus lipase (Aspergillus fumigatus lip2,SEQ ID NOs: 3 and 4, respectively).

FIG. 9 shows a restriction map of pHyGe026.

FIGS. 10A and 10B show the genomic DNA sequence and the deduced aminoacid sequence of a Magnaporthe grisea lipase (Magnaporthe grisea lip1,SEQ ID NOs: 5 and 6, respectively).

FIG. 11 shows a restriction map of pHyGe010.

FIG. 12 shows a restriction map of pCrAm138.

FIG. 13 shows the genomic DNA sequence and the deduced amino acidsequence of a Magnaporthe grisea lipase (Magnaporthe grisea lip2, SEQ IDNOs: 7 and 8, respectively).

FIG. 14 shows a restriction map of pBM135g.

FIG. 15 shows the genomic DNA sequence and the deduced amino acidsequence of a Magnaporthe grisea lipase (Magnaporthe grisea lip3, SEQ IDNOs: 9 and 10, respectively).

FIG. 16 shows a restriction map of pJLin171.

FIG. 17 shows the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus nidulans lipase (Aspergillus nidulans lip1,SEQ ID NOs: 11 and 12, respectively).

FIG. 18 shows a restriction map of pJLin167.

FIG. 19 shows a restriction map of pJLin170.

FIG. 20 shows the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus nidulans lipase (Aspergillus nidulans lip2,SEQ ID NOs: 13 and 14, respectively).

FIG. 21 shows a restriction map of pJSF8c.

FIG. 22 shows a restriction map of pBM141.

FIG. 23 shows the genomic DNA sequence and the deduced amino acidsequence of an Aspergillus nidulans lipase (Aspergillus nidulans lip3,SEQ ID NOs: 15 and 16, respectively).

DEFINITIONS

Lipase activity: The term “lipase activity” is defined herein as atriacylglycerol acylhydrolase activity (E.C. 3.1.1.3) which catalyzesthe hydrolysis of a triacylglycerol to fatty acid(s). The substratespectrum of lipases includes triglycerides, diglycerides, andmonoglycerides, but for the purpose of the present invention, lipaseactivity is determined using p-nitrophenyl butyrate as substrate. Oneunit of lipase activity equals the amount of enzyme capable of releasing1 μmole of butyric acid per minute at pH 7.5, 25° C. Encompassed withinthe term “lipase activity” are polypeptides that also have phopholipaseactivity and/or galactolipase activity, as defined herein.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the lipase activity of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID. NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16.

Phospholipase activity: The term “phospholipase activity” is definedherein as a phosphatidyl acylhydrolase (EC 3.1.1.4, EC 3.1.1.5, and EC3.1.1.32) which catalyzes the hydrolysis of a fatty acid from aphospholipid. Phospholipids consist of a glycerol backbone with twofatty acids in the sn1 and sn2 positions, which is esterified with aphosphoric acid in the sn3 position. The phosphoric acid may, in turn,be esterified with an amino alcohol.

Phospholipase A₁ (EC 3.1.1.32) catalyzes the deacylation of one fattyacyl group in the sn1 position from a diacylglycerophospholipid toproduce lysophospholipid and fatty acid.

Phospholipase A₂ (EC 3.1.1.4) catalyzes the deacylation of one fattyacyl group in the sn2 position from a diacylglycerophospholipid toproduce lysophospholipid and fatty acid.

Lysophospholipase (EC 3.1.1.5), also known as phospholipase B, catalyzesthe hydrolysis of the fatty acyl group in a lysophospholipid.

For purposes of the present invention, phospholipase A₁, phospholipaseA₂, and lysophospholipase activity is determined according to WO2005/040410 using phosphatidylcholines derived from soy (Avanti PolarLipids Inc., AL, USA) as substrate.

Galactolipase Activity: The term “galactolipase activity” is definedherein as a 1,2-diacyl-3-beta-D-galactosyl-sn-glycerol acylhydrolase (EC3.1.1.26) which catalyzes the hydrolysis of galactolipids by removingone or two fatty acids. Galactolipase activity is determined accordingto WO 2005/040410 using digalactosyldiglyceride (DGDG) ormonogalactosyldiglyceride (MGDG) extracted from wheat flour assubstrate. DGDG is a galactolipid that consists of two fatty acids and adigalactose. MGDG is a galactolipid that consists of two fatty acids anda galactose.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively associated. It is, therefore,preferred that the substantially pure polypeptide is at least 92% pure,preferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 96% pure, morepreferably at least 97% pure, more preferably at least 98% pure, evenmore preferably at least 99%, most preferably at least 99.5% pure, andeven most preferably 100% pure by weight of the total polypeptidematerial present in the preparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively associated. This can be accomplished, for example, bypreparing the polypeptide by means of well-known recombinant methods orby classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having lipase activity that is in its final form followingtranslation and any post-translational modifications, such as N-terminalprocessing, C-terminal truncation, glycosylation, etc.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined by the Clustal method (Higgins,1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software(DNASTAR, Inc., Madison, Wis.) with an identity table and the followingmultiple alignment parameters: Gap penalty of 10 and gap length penaltyof 10. Pairwise alignment parameters are Ktuple=1, gap penalty=3,windows=5, and diagonals=5.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein which gives an E value (or expectancy score) of lessthan 0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Thermomyces lanuginosus lipase (Accession No. 059952).

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, or SEQ ID NO: 16; or a homologous sequence thereof; whereinthe fragment has lipase activity. In a preferred aspect, a fragmentcontains at least 325 amino acid residues, more preferably at least 340amino acid residues, and most preferably at least 355 amino acidresidues of the mature polypeptide of SEQ ID NO: 2 or a homologoussequence thereof. In another preferred aspect, a fragment contains atleast 215 amino acid residues, more preferably at least 230 amino acidresidues, and most preferably at least 245 amino acid residues of themature polypeptide of SEQ ID NO: 4 or a homologous sequence thereof. Inanother preferred aspect, a fragment contains at least 255 amino acidresidues, more preferably at least 270 amino acid residues, and mostpreferably at least 285 amino acid residues of the mature polypeptide ofSEQ ID NO: 6 or a homologous sequence thereof. In another preferredaspect, a fragment contains at least 285 amino acid residues, morepreferably at least 300 amino acid residues, and most preferably atleast 315 amino acid residues of the mature polypeptide of SEQ ID NO: 8or a homologous sequence thereof. In another preferred aspect, afragment contains at least 320 amino acid residues, more preferably atleast 335 amino acid residues, and most preferably at least 350 aminoacid residues of the mature polypeptide of SEQ ID NO: 10 or a homologoussequence thereof. In another preferred aspect, a fragment contains atleast 230 amino acid residues, more preferably at least 245 amino acidresidues, and most preferably at least 260 amino acid residues of themature polypeptide of SEQ ID NO: 12 or a homologous sequence thereof. Inanother preferred aspect, a fragment contains at least 240 amino acidresidues, more preferably at least 255 amino acid residues, and mostpreferably at least 270 amino acid residues of the mature polypeptide ofSEQ ID NO: 14 or a homologous sequence thereof. In another preferredaspect, a fragment contains at least 320 amino acid residues, morepreferably at least 340 amino acid residues, and most preferably atleast 360 amino acid residues of the mature polypeptide of SEQ ID NO: 16or a homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, or SEQ ID NO: 15; or a homologous sequence thereof; wherein thesubsequence encodes a polypeptide fragment having lipase activity. In apreferred aspect, a subsequence contains at least 975 nucleotides, morepreferably at least 1020 nucleotides, and most preferably at least 1060nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 ora homologous sequence thereof. In another preferred aspect, asubsequence contains at least 645 nucleotides, more preferably at least690 nucleotides, and most preferably at least 735 nucleotides of themature polypeptide coding sequence of SEQ ID NO: 3 or a homologoussequence thereof. In a preferred aspect, a subsequence contains at least765 nucleotides, more preferably at least 810 nucleotides, and mostpreferably at least 855 nucleotides of the mature polypeptide codingsequence of SEQ ID NO: 5 or a homologous sequence thereof. In anotherpreferred aspect, a subsequence contains at least 855 nucleotides, morepreferably at least 900 nucleotides, and most preferably at least 945nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 7 ora homologous sequence thereof. In another preferred aspect, asubsequence contains at least 960 nucleotides, more preferably at least1005 nucleotides, and most preferably at least 1050 nucleotides of themature polypeptide coding sequence of SEQ ID NO: 9 or a homologoussequence thereof. In a preferred aspect, a subsequence contains at least690 nucleotides, more preferably at least 735 nucleotides, and mostpreferably at least 780 nucleotides of the mature polypeptide codingsequence of SEQ ID NO: 11 or a homologous sequence thereof. In anotherpreferred aspect, a subsequence contains at least 720 nucleotides, morepreferably at least 765 nucleotides, and most preferably at least 810nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 13or a homologous sequence thereof. In another preferred aspect, asubsequence contains at least 1020 nucleotides, more preferably at least1020 nucleotides, and most preferably at least 1080 nucleotides of themature polypeptide coding sequence of SEQ ID NO: 15 or a homologoussequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 2⁰% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively associated. A substantially pure polynucleotide may, however,include naturally occurring 5′ and 3′ untranslated regions, such aspromoters and terminators. It is preferred that the substantially purepolynucleotide is at least 90% pure, preferably at least 92% pure, morepreferably at least 94% pure, more preferably at least 95% pure, morepreferably at least 96% pure, more preferably at least 97% pure, evenmore preferably at least 98% pure, most preferably at least 99%, andeven most preferably at least 99.5% pure by weight. The polynucleotidesof the present invention are preferably in a substantially pure form. Inparticular, it is preferred that the polynucleotides disclosed hereinare in “essentially pure form”, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively associated. Herein, the term “substantially purepolynucleotide” is synonymous with the terms “isolated polynucleotide”and “polynucleotide in isolated form.” The polynucleotides may be ofgenomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinationsthereof.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having lipase activity.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG and TGA. The coding sequence may be aDNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; or a homologous sequencethereof; as well as genetic manipulation of the DNA encoding such apolypeptide. The modification can be substitutions, deletions and/orinsertions of one or more amino acids as well as replacements of one ormore amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having lipase activity produced by an organismexpressing a modified nucleotide sequence of the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; or ahomologous sequence thereof. The modified nucleotide sequence isobtained through human intervention by modification of the nucleotidesequence disclosed in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; ora homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Lipase Activity

In a first aspect, the present invention relates to isolatedpolypeptides having an amino acid sequence which has a degree ofidentity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQID NO: 16, of at least 60%, preferably at least 65%, more preferably atleast 70%, more preferably at least 75%, more preferably at least 80%,more preferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 97%, 98%, or99%, which have lipase activity (hereinafter “homologous polypeptides”).In a preferred aspect, the homologous polypeptides have an amino acidsequence which differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, or SEQ ID NO: 16.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 2. In another preferred aspect, a polypeptide comprisesamino acids 25 to 396 of SEQ ID NO: 2, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 25 to 396 of SEQ ID NO: 2.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, a polypeptideconsists of amino acids 25 to 396 of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has lipase activity. In anotherpreferred aspect, a polypeptide consists of amino acids 25 to 396 of SEQID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 4, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 4. In another preferred aspect, a polypeptide comprisesamino acids 25 to 283 of SEQ ID NO: 4, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 25 to 283 of SEQ ID NO: 4.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide consists of amino acids 25 to283 of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereofthat has lipase activity. In another preferred aspect, a polypeptideconsists of amino acids 25 to 283 of SEQ ID NO: 4.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 6, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 6. In another preferred aspect, a polypeptide comprisesamino acids 20 to 318 of SEQ ID NO: 6, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 20 to 318 of SEQ ID NO: 6.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 6 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide consists of amino acids 20 to318 of SEQ ID NO: 6 or an allelic variant thereof; or a fragment thereofthat has lipase activity. In another preferred aspect, a polypeptideconsists of amino acids 20 to 318 of SEQ ID NO: 6.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 8, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 8. In another preferred aspect, a polypeptide comprisesamino acids 19 to 348 of SEQ ID NO: 8, or an allelic variant thereof; ora fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 19 to 348 of SEQ ID NO: 8.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 8 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, a polypeptide consists of amino acids 19 to348 of SEQ ID NO: 8 or an allelic variant thereof; or a fragment thereofthat has lipase activity. In another preferred aspect, a polypeptideconsists of amino acids 19 to 348 of SEQ ID NO: 8.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 10, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 10. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 10. In another preferred aspect, a polypeptide comprisesamino acids 25 to 393 of SEQ ID NO: 10, or an allelic variant thereof;or a fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 25 to 393 of SEQ ID NO: 10.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 10 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 10. Inanother preferred aspect, a polypeptide consists of amino acids 25 to393 of SEQ ID NO: 10 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of amino acids 25 to 393 of SEQ ID NO: 10.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 12, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 12. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 12. In another preferred aspect, a polypeptide comprisesamino acids 20 to 294 of SEQ ID NO: 12, or an allelic variant thereof;or a fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 20 to 294 of SEQ ID NO: 12.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 12 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 12. Inanother preferred aspect, a polypeptide consists of amino acids 20 to294 of SEQ ID NO: 12 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of amino acids 20 to 294 of SEQ ID NO: 12.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 14, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 14. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 14. In another preferred aspect, a polypeptide comprisesamino acids 25 to 308 of SEQ ID NO: 14, or an allelic variant thereof;or a fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 25 to 308 of SEQ ID NO: 14.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 14 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 14. Inanother preferred aspect, a polypeptide consists of amino acids 25 to308 of SEQ ID NO: 14 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of amino acids 25 to 308 of SEQ ID NO: 14.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 16, or an allelic variant thereof; or afragment thereof that has lipase activity. In a preferred aspect, apolypeptide comprises the amino acid sequence of SEQ ID NO: 16. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 16. In another preferred aspect, a polypeptide comprisesamino acids 26 to 404 of SEQ ID NO: 16, or an allelic variant thereof;or a fragment thereof that has lipase activity. In another preferredaspect, a polypeptide comprises amino acids 0.26 to 404 of SEQ ID NO:16. In another preferred aspect, a polypeptide consists of the aminoacid sequence of SEQ ID NO: 16 or an allelic variant thereof; or afragment thereof that has lipase activity. In another preferred aspect,a polypeptide consists of the amino acid sequence of SEQ ID NO: 16. Inanother preferred aspect, a polypeptide consists of amino acids 26 to404 of SEQ ID NO: 16 or an allelic variant thereof; or a fragmentthereof that has lipase activity. In another preferred aspect, apolypeptide consists of amino acids 26 to 404 of SEQ ID NO: 16.

In a second aspect, the present invention relates to isolatedpolypeptides having lipase activity which are encoded by polynucleotideswhich hybridize under very low stringency conditions, preferably lowstringency conditions, more preferably medium stringency conditions,more preferably medium-high stringency conditions, even more preferablyhigh stringency conditions, and most preferably very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, or SEQ ID NO: 15, (ii) the cDNA sequence contained inthe mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,or SEQ ID NO: 15, (iii) a subsequence of (i) or (ii), or (iv) acomplementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2dedition, Cold Spring Harbor, N.Y.). A subsequence of the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:15 contains at least 100 contiguous nucleotides or preferably at least200 contiguous nucleotides. Moreover, the subsequence may encode apolypeptide fragment which has lipase activity. In a preferred aspect,the mature polypeptide coding sequence is nucleotides 73 to 1256 of SEQID NO: 1, nucleotides 73 to 944 of SEQ ID NO: 3, nucleotides 58 to 1085of SEQ ID NO: 5, nucleotides 55 to 1044 of SEQ ID NO: 7, nucleotides 73to 1179 of SEQ ID NO: 9, nucleotides 58 to 1038 of SEQ ID NO: 11,nucleotides 73 to 1116 of SEQ ID NO: 13, or nucleotides 76 to 1280 ofSEQ ID NO: 15.

The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15;or a subsequence thereof; as well as the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; or a fragment thereof; may beused to design a nucleic acid probe to identify and clone DNA encodingpolypeptides having lipase activity from strains of different genera orspecies according to methods well known in the art. In particular, suchprobes can be used for hybridization with the genomic or cDNA of thegenus or species of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 14, preferably at least 25, morepreferably at least 35, and most preferably at least 70 nucleotides inlength. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes which are at least 600 nucleotides, at least preferably atleast 700 nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having lipase activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15; or asubsequence thereof; the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, or SEQ ID NO: 15, the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQID NO: 15; its complementary strand; or a subsequence thereof; undervery low to very high stringency conditions. Molecules to which thenucleic acid probe hybridizes under these conditions can be detectedusing X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 73 to 1256 of SEQ ID NO: 1. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencewhich encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pSMO224 which is contained in E. coli NRRLB-30774, wherein the polynucleotide sequence thereof encodes apolypeptide having lipase activity. In another preferred aspect, thenucleic acid probe is the mature polypeptide coding region contained inplasmid pSMO224 which is contained in E. coli NRRL B-30774.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 944 of SEQ ID NO: 3.In another preferred aspect, the nucleic acid probe is a polynucleotidesequence which encodes the polypeptide of SEQ ID NO: 4, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 3. In another preferred aspect, the nucleic acid probe is thepolynucleotide sequence contained in plasmid pSMO223 which is containedin E. coli NRRL B-30773, wherein the polynucleotide sequence thereofencodes a polypeptide having lipase activity. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding regioncontained in plasmid pSMO223 which is contained in E. coli NRRL B-30773.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1085 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 6,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pHyGe026 whichis contained in E. coli NRRL B-30772, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pHyGe026 which iscontained in E. coli NRRL B-30772.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 55 to 1044 of SEQ ID NO:7. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 8,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 7. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pCrAm138 whichis contained in E. coli NRRL B-30781, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pCrAm138 which iscontained in E. coli NRRL B-30781.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 1179 of SEQ ID NO:9. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 10,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 9. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pBM135g whichis contained in E. coli NRRL B-30779, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pBM135g which iscontained in E. coli NRRL B-30779.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 11. In another preferredaspect, the nucleic acid probe is nucleotides 58 to 1038 of SEQ ID NO:11. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 12,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 11. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pJLin171 whichis contained in E. coli NRRL B-30755, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pJLin171 which iscontained in E. coli NRRL B-30755.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 13. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 1116 of SEQ ID NO:13. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 18,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 13. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pJLin170 whichis contained in E. coli NRRL B-30754, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pJLin170 which iscontained in E. coli NRRL B-30754.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 15. In another preferredaspect, the nucleic acid probe is nucleotides 76 to 1280 of SEQ ID NO:15. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 16,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 15. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pBM141 whichis contained in E. coli NRRL B-30780, wherein the polynucleotidesequence thereof encodes a polypeptide having lipase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pBM141 which is containedin E. coli NRRL B-30780.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, or SEQ ID NO: 16; or a homologous sequence thereof.Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,lipase activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The active site of the enzyme or other biologicalinteraction can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction, or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. See, forexample, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992,J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred fromanalysis of identities with polypeptides which are related to apolypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, such as aminoacids 25 to 396 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 4, such as aminoacids 25 to 283 of SEQ ID NO: 4, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 6, such as aminoacids 20 to 318 of SEQ ID NO: 6, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 8, such as aminoacids 19 to 348 of SEQ ID NO: 8, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 10, such as aminoacids 25 to 393 of SEQ ID NO: 10, is 10, preferably 9, more preferably8, more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 12, such as aminoacids 20 to 294 of SEQ ID NO: 12, is 10, preferably 9, more preferably8, more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 14, such as aminoacids 25 to 308 of SEQ ID NO: 14, is 10, preferably 9, more preferably8, more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 16, such as aminoacids 26 to 404 of SEQ ID NO: 16, is 10, preferably 9, more preferably8, more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

Sources of Polypeptides Having Lipase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus polypeptide having lipase activity, e.g.,a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, or Bacillus thuringiensispolypeptide having lipase activity; or a Streptomyces polypeptide havinglipase activity, e.g., a Streptomyces lividans or Streptomyces murinuspolypeptide having lipase activity; or a gram negative bacterialpolypeptide having lipase activity, e.g., an E. coli or a Pseudomonassp. polypeptide having lipase activity.

A polypeptide of the present invention may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide; or more preferably a filamentous fungal polypeptide such asan Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium,Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocalimastix,Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum,Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichodermapolypeptide having lipase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having lipaseactivity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioldes, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Mycellophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride polypeptide having lipaseactivity.

In another preferred aspect, the polypeptide is a Thielavia achromatica,Thielavia albomyces, Thlelavia albopilosa, Thielavia australelnsis,Thielavla fimeti, Thielavia microspora, Thielavia ovlspora, Thielavlaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophlla, Thielavia terrestris, Thielavia terricola, Thielaviathermophila, Thielavia variospora, or Thielavia wareingii polypeptidehaving lipase activity.

In a more preferred aspect, the polypeptide is an Aspergillus fumigatuspolypeptide, e.g., the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, orthe mature polypeptide thereof.

In another more preferred aspect, the polypeptide is a Magnaporthegrisea polypeptide, and most preferably a Magnaporthe grisea FGSC 8958(Fungal Genetics Stock Center, Kansas City, Mo.) polypeptide, e.g., thepolypeptide of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or themature polypeptide thereof.

In another more preferred aspect, the polypeptide is an Aspergillusnidulans polypeptide, and most preferably an Aspergillus nidulans A1000(Fungal Genetics Stock Center, Kansas City, Mo.) polypeptide, e.g., thepolypeptide of SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, or themature polypeptide thereof.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having anucleotide sequence which encode a polypeptide of the present invention.

In a preferred aspect, the nucleotide sequence is set forth in SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence is thesequence contained in plasmid pSMO224 which is contained in E. coli NRRLB-30774. In another preferred aspect, the nucleotide sequence is themature polypeptide coding region of SEQ ID NO: 1. In another preferredaspect, the nucleotide sequence is nucleotides 73 to 1256 of SEQ IDNO: 1. In another more preferred aspect, the nucleotide sequence is themature polypeptide coding region contained in plasmid pSMO224 which iscontained in E. coli NRRL B-30774. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof,which differ from SEQ ID NO: 1 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 1 which encodefragments of SEQ ID NO: 2 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 3. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pSMO223 which is contained in E. coliNRRL B-30773. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 3. In anotherpreferred aspect, the nucleotide sequence is nucleotides 73 to 944 ofSEQ ID NO: 3. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pSMO223which is contained in E. coli NRRL B-30773. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 4 or the mature polypeptide thereof,which differ from SEQ ID NO: 3 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 3 which encodefragments of SEQ ID NO: 4 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 5. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pHyGe026 which is contained in E. coliNRRL B-30772. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 5. In anotherpreferred aspect, the nucleotide sequence is nucleotides 58 to 1085 ofSEQ ID NO: 5. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pHyGe026which is contained in E. coli NRRL B-30772. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 6 or the mature polypeptide thereof,which differ from SEQ ID NO: 5 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 5 which encodefragments of SEQ ID NO: 6 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 7. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pCrAm138 which is contained in E. coliNRRL B-30781. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 7. In anotherpreferred aspect, the nucleotide sequence is nucleotides 55 to 1044 ofSEQ ID NO: 7. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pCrAm138which is contained in E. coli NRRL B-30781. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 8 or the mature polypeptide thereof,which differ from SEQ ID NO: 7 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 7 which encodefragments of SEQ ID NO: 8 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 9. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pBM135g which is contained in E. coliNRRL B-30779. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 9. In anotherpreferred aspect, the nucleotide sequence is nucleotides 73 to 1179 ofSEQ ID NO: 9. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pBM135gwhich is contained in E. coli NRRL B-30779. The present invention alsoencompasses nucleotide sequences which encode a polypeptide having theamino acid sequence of SEQ ID NO: 10 or the mature polypeptide thereof,which differ from SEQ ID NO: 9 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 9 which encodefragments of SEQ ID NO: 10 that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 11. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pJLin171 which is contained in E. coliNRRL B-30755. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 11. In anotherpreferred aspect, the nucleotide sequence is nucleotides 58 to 1038 ofSEQ ID NO: 11. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pJLin171which is contained in E. coli NRRL B-30755. In another more preferredaspect, the nucleotide sequence is the mature polypeptide coding regioncontained in plasmid pJLin171 which is contained in E. coli NRRLB-30755. The present invention also encompasses nucleotide sequenceswhich encode a polypeptide having the amino acid sequence of SEQ ID NO:12 or the mature polypeptide thereof, which differ from SEQ ID NO: 11 orthe mature polypeptide coding sequence thereof by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 11 which encode fragments of SEQ ID NO: 12that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 13. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pJLin170 which is contained in E. coliNRRL B-30754. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 13. In anotherpreferred aspect, the nucleotide sequence is nucleotides 73 to 1116 ofSEQ ID NO: 13. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pJLin170which is contained in E. coli NRRL B-30754. In another more preferredaspect, the nucleotide sequence is the mature polypeptide coding regioncontained in plasmid pJLin170 which is contained in E. coli NRRLB-30754. The present invention also encompasses nucleotide sequenceswhich encode a polypeptide having the amino acid sequence of SEQ ID NO:14 or the mature polypeptide thereof, which differ from SEQ ID NO: 13 orthe mature polypeptide coding sequence thereof by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 13 which encode fragments of SEQ ID NO: 14that have lipase activity.

In another preferred aspect, the nucleotide sequence is set forth in SEQID NO: 15. In another more preferred aspect, the nucleotide sequence isthe sequence contained in plasmid pBM141 which is contained in E. coliNRRL B-30780. In another preferred aspect, the nucleotide sequence isthe mature polypeptide coding region of SEQ ID NO: 15. In anotherpreferred aspect, the nucleotide sequence is nucleotides 76 to 1280 ofSEQ ID NO: 15. In another more preferred aspect, the nucleotide sequenceis the mature polypeptide coding region contained in plasmid pBM141which is contained in E. coli NRRL B-30780. In another more preferredaspect, the nucleotide sequence is the mature polypeptide coding regioncontained in plasmid pBM141 which is contained in E. coli NRRL B-30780.The present invention also encompasses nucleotide sequences which encodea polypeptide having the amino acid sequence of SEQ ID NO: 16 or themature polypeptide thereof, which differ from SEQ ID NO: 15 or themature polypeptide coding sequence thereof by virtue of the degeneracyof the genetic code. The present invention also relates to subsequencesof SEQ ID NO: 15 which encode fragments of SEQ ID NO: 16 that havelipase activity.

The present invention also relates to mutant polunucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 2. In a preferred aspect, the maturepolypeptide is amino acids 25 to 396 of SEQ ID NO: 2.

The present invention also relates to mutant polunucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 3, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 4. In a preferred aspect, the maturepolypeptide is amino acids 25 to 283 of SEQ ID NO: 4.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 5, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 6. In a preferred aspect, the maturepolypeptide is amino acids 20 to 318 of SEQ ID NO: 6.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 7, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 8. In a preferred aspect, the maturepolypeptide is amino acids 19 to 348 of SEQ ID NO: 8.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 9, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 10. In a preferred aspect, the maturepolypeptide is amino acids 25 to 393 of SEQ ID NO: 10.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 11, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 12. In a preferred aspect, the maturepolypeptide is amino acids 20 to 294 of SEQ ID NO: 12.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 13, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 14. In a preferred aspect, the maturepolypeptide is amino acids 25 to 308 of SEQ ID NO: 14.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 15, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 16. In a preferred aspect, the maturepolypeptide is amino acids 26 to 404 of SEQ ID NO: 16.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Thielavia, or another or related organismand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the nucleotide sequence.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 of at least 60%, preferably at least65%, more preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, even more preferably at least 95%, and most preferably atleast 97% identity, which encode an active polypeptide. In a preferredaspect, the mature polypeptide coding sequence is nucleotides 73 to 1256of SEQ ID NO: 1.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 3 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 73 to 944 of SEQ ID NO: 3.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 5 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 58 to 1085 of SEQ ID NO: 5.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 7 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 55 to 1044 of SEQ ID NO: 7.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 9 of at least 70%, preferably at least75%, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 73 to 1179 of SEQ ID NO: 9.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 11 of at least 70%, preferably at least75%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 95%, and most preferably at least 97% identity,which encode an active polypeptide. In a preferred aspect, the maturepolypeptide coding sequence is nucleotides 58 to 1038 of SEQ ID NO: 11.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 13 of at least 70%, preferably at least75%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 95%, and most preferably at least 97% identity,which encode an active polypeptide. In a preferred aspect, the maturepolypeptide coding sequence is nucleotides 73 to 1116 of SEQ ID NO: 13.

The present invention also relates to polynucleotides having nucleotidesequences which have a degree of identity to the mature polypeptidecoding sequence of SEQ ID NO: 15 of at least 70%, preferably at least75%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, even morepreferably at least 95%, and most preferably at least 97% identity,which encode an active polypeptide. In a preferred aspect, the maturepolypeptide coding sequence is nucleotides 76 to 1280 of SEQ ID NO: 15.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, e.g., a subsequence thereof,and/or by introduction of nucleotide substitutions which do not giverise to another amino acid sequence of the polypeptide encoded by thenucleotide sequence, but which correspond to the codon usage of the hostorganism intended for production of the enzyme, or by introduction ofnucleotide substitutions which may give rise to a different amino acidsequence. For a general description of nucleotide substitution, see,e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, Science 244: 1081-1085). In the lattertechnique, mutations are introduced at every positively charged residuein the molecule, and the resultant mutant molecules are tested forlipase activity to identify amino acid residues that are critical to theactivity of the molecule. Sites of substrate-enzyme interaction can alsobe determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO:15, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or (iii) acomplementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.In a preferred aspect, the mature polypeptide coding sequence of SEQ IDNO: 1 is nucleotides 73 to 1259. In another preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 3 is nucleotides 73 to 947. Inanother preferred aspect, the mature polypeptide coding sequence of SEQID NO: 5 is nucleotides 58 to 1088. In another preferred aspect, themature polypeptide coding sequence of SEQ ID NO: 7 is nucleotides 55 to1044. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 9 is nucleotides 73 to 1179. In another preferredaspect, the mature polypeptide coding sequence of SEQ ID NO: 11 isnucleotides 58 to 1041. In another preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 13 is nucleotides 73 to 1119.In another preferred aspect, the mature polypeptide coding sequence ofSEQ ID NO: 15 is nucleotides 76 to 1280.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQID NO: 15, (ii) the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, or(iii) a complementary strand of (i) or (ii); and (b) isolating thehybridizing polynucleotide, which encodes a polypeptide having lipaseactivity. In a preferred aspect, the mature polypeptide coding sequenceof SEQ ID NO: 1 is nucleotides 73 to 1259. In another preferred aspect,the mature polypeptide coding sequence of SEQ ID NO: 3 is nucleotides 73to 947. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 5 is nucleotides 58 to 1088. In another preferredaspect, the mature polypeptide coding sequence of SEQ ID NO: 7 isnucleotides 55 to 1044. In another preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 9 is nucleotides 73 to 1179.In another preferred aspect, the mature polypeptide coding sequence ofSEQ ID NO: 11 is nucleotides 58 to 1041. In another preferred aspect,the mature polypeptide coding sequence of SEQ ID NO: 13 is nucleotides73 to 1119. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 15 is nucleotides 76 to 1280.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusnigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide coding region is nucleotides 1to 72 of SEQ ID NO: 1 which encode amino acids 1 to 24 of SEQ ID NO: 2.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 72 of SEQ ID NO: 3 which encode amino acids 1 to 24 ofSEQ ID NO: 4.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 42 of SEQ ID NO: 5 which encode amino acids 1 to 19 ofSEQ ID NO: 6.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 54 of SEQ ID NO: 7 which encode amino acids 1 to 18 ofSEQ ID NO: 8.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 72 of SEQ ID NO: 9 which encode amino acids 1 to 24 ofSEQ ID NO: 10.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 57 of SEQ ID NO: 11 which encode amino acids 1 to 19 ofSEQ ID NO: 12.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 72 of SEQ ID NO: 13 which encode amino acids 1 to 24 ofSEQ ID NO: 14.

In another preferred aspect, the signal peptide coding region isnucleotides 1 to 75 of SEQ ID NO: 15 which encode amino acids 1 to 25 ofSEQ ID NO: 16.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GALL systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus otyzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis, or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Fillbasidium, Fusarium, Humicola,Magnaporthe, Mucor, Mycellophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioldes, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Mycellophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known perse. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. In a preferred aspect,the cell is of the genus Aspergillus. In a more preferred aspect, thecell is Aspergillus fumigatus. In another more preferred aspect, thecell is Aspergillus nidulans. In another preferred aspect, the cell isof the genus Magnaporthe. In another more preferred aspect, the cell isMagnaporthe grisea.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, or SEQ ID NO: 15, wherein the mutant nucleotidesequence encodes a polypeptide which comprises or consists of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16, and (b)recovering the polypeptide.

In a preferred aspect, the mature polypeptide of SEQ ID NO: 2 is aminoacids 25 to 396. In another preferred aspect, the mature polypeptide ofSEQ ID NO: 4 is amino acids 25 to 283. In another preferred aspect, themature polypeptide of SEQ ID NO: 6 is amino acids 20 to 318. In anotherpreferred aspect, the mature polypeptide of SEQ ID NO: 8 is amino acids19 to 348. In another preferred aspect, the mature polypeptide of SEQ IDNO: 10 is amino acids 25 to 393. In another preferred aspect, the maturepolypeptide of SEQ ID NO: 12 is amino acids 20 to 294. In anotherpreferred aspect, the mature polypeptide of SEQ ID NO: 14 is amino acids25 to 308. In another preferred aspect, the mature polypeptide of SEQ IDNO: 16 is amino acids 26 to 404.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having lipase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome or chloroplastgenome and propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having lipase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Lipase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising: (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of lipase activity byfermentation of a cell which produces both a polypeptide of the presentinvention as well as the protein product of interest by adding aneffective amount of an agent capable of inhibiting lipase activity tothe fermentation broth before, during, or after the fermentation hasbeen completed, recovering the product of interest from the fermentationbroth, and optionally subjecting the recovered product to furtherpurification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of lipase activity bycultivating the cell under conditions permitting the expression of theproduct, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the lipase activity substantially,and recovering the product from the culture broth. Alternatively, thecombined pH and temperature treatment may be performed on an enzymepreparation recovered from the culture broth. The combined pH andtemperature treatment may optionally be used in combination with atreatment with an lipase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the lipase activity. Complete removal of lipase activity may beobtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallylipase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The enzyme may be selected from, e.g., an amylolytic enzyme, lipolyticenzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plantcell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Thelipase-deficient cells may also be used to express heterologous proteinsof pharmaceutical interest such as hormones, growth factors, receptors,and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from lipase activity which is produced by a method ofthe present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thelipase activity of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae, Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having lipase activity, or compositions thereof.

Use in Degumming. A polypeptide of the present invention may be used fordegumming an aqueous carbohydrate solution or slurry to improve itsfilterability, particularly, a starch hydrolysate, especially a wheatstarch hydrolysate which is difficult to filter and yields cloudyfiltrates. The treatment may be performed using methods well known inthe art. See, for example, EP 219,269, EP 808,903, and U.S. Pat. No.6,103,505.

Use in Baking. A polypeptide of the present invention may be used inbaking according to U.S. Pat. No. 6,558,715.

Use in detergent. The polypeptides of the present invention may be addedto and thus become a component of a detergent composition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the present invention as describedherein. The detergent additive as well as the detergent composition maycomprise one or more enzymes such as a protease, lipase, cutinase, anamylase, carbohydrase, cellulase, pectinase, mannanase, arabinase,galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™, Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta,1131: 253-360), B. stearothermophilus(JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include Lipolase™,Lipolase Ultra™, and Lipex™ (Novozymes A/S).

Amylases: Suitable amylases (α and/or β) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, α-amylases obtained fromBacillus, e.g., a special strain of Bacillus licheniformis, described inmore detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

In the detergent compositions, a polypeptide of the present inventionmay be added in an amount corresponding to 0.001-100 mg of protein,preferably 0.005-50 mg of protein, more preferably 0.01-25 mg ofprotein, even more preferably 0.05-10 mg of protein, most preferably0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein perliter of wash liquor.

A polypeptide of the present invention may also be incorporated in thedetergent formulations disclosed in WO 97/07202, which is herebyincorporated by reference.

Signal Peptide

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to a nucleotide sequencecomprising or consisting of nucleotides 1 to 72 of SEQ ID NO: 1,nucleotides 1 to 72 of SEQ ID NO: 3, nucleotides 1 to 57 of SEQ ID NO:5, nucleotides 1 to 54 of SEQ ID NO: 7, nucleotides 1 to 72 of SEQ IDNO: 9, nucleotides 1 to 57 of SEQ ID NO: 11, nucleotides 1 to 72 of SEQID NO: 13, or nucleotides 1 to 75 of SEQ ID NO: 15 encoding a signalpeptide comprising or consisting of amino acids 1 to 24 of SEQ ID NO: 2,amino acids 1 to 24 of SEQ ID NO: 4, amino acids 1 to 19 of SEQ ID NO:6, amino acids 1 to 18 of SEQ ID NO: 8, amino acids 1 to 24 of SEQ IDNO: 10, amino acids 1 to 19 of SEQ ID NO: 12, amino acids 1 to 24 of SEQID NO: 14, or amino acids 1 to 25 of SEQ ID NO: 16, respectively,wherein the gene is foreign to the nucleotide sequence.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Aspergillus fumigatus PaHa34, Magnaporthe grisea FGSC 8958 (FungalGenetics Stock Center), and Aspergillus nidulans A1000 (Fungal GeneticsStock Center, Kansas City, Mo.) were used as sources for the lipasegenes.

Media

Potato dextrose medium was composed per liter of 24 grams of potatodextrose.

YP medium was composed per liter of 10 g of yeast extract and 20 g ofBacto peptone.

SOC medium was composed per liter of 20 g of tryptone, 5 g of yeastextract, 2 ml of 5 M NaCl, and 2.5 ml of 1 M KCl.

NZY⁺ medium was composed per liter of 10 g of NZ amine, 5 g of yeastextract, 5 g of NaCl, 12.5 mM MgCl₂, 12.5 mM MgSO₄, and 10 ml of 2 Mglucose.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of NaCl.

MY25 medium was composed per liter of 25 g of maltodextrin, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of citric acid, 2 g of K₂SO₄, 2 g ofurea, 10 g of yeast extract, and 1.5 ml of AMG trace metals solution,adjusted to pH 6.

AMG trace metals solution was composed per liter of 14.3 g ofZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g ofFeSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, and 3 g of citric acid.

CM was composed per liter of 6 g of yeast extract, 6 g of casein acidhydrolysate, and 10 g of sucrose.

COVE selection plates were composed per liter of 342.3 g of sucrose, 20ml of COVE salt solution, 10 mM acetamide, 15 mM CsCl₂, and 25 g ofNoble agar.

COVE salt solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals solution.

COVE trace metals solution was composed per liter of 0.04 g ofNaB₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

2×YT plates were composed per liter of 16 g of tryptone, 10 g of yeastextract, 5 g of NaCl, and 15 g of Bacto agar.

Example 1 Identification of Lipase Genes in the Partial Genomic Sequenceof Aspergillus fumigatus

A tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods andProtocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) of theAspergillus fumigatus partial genome sequence (The Institute for GenomicResearch, Rockville, Md.) was carried out using as query a lipasesequence from Thermomyces lanuginosus (Accession No. 059952). Severalgenes were identified as putative lipases based upon an E value of lessthan 0.001 in the tfasty output. Two genomic regions of approximately900 bp and 570 bp with greater than 33% identity to the query sequenceat the amino acid level were chosen for further study. The gene modelsfor the putative lipase genes were predicted based on homology to theThermomyces lanuginosus lipase as well as conserved sequences present atthe 5′ and 3′ ends of fungal introns.

Example 2 Aspergillus fumigatus Genomic DNA Extraction

Aspergillus fumigatus was grown in 250 ml of potato dextrose medium in abaffled shake flask at 37° C. and 240 rpm. Mycelia were harvested byfiltration, washed twice in TE (10 mM Tris-1 mM EDTA), and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, which was resuspended in pH 8.0 buffer containing 10 mMTris, 100 mM EDTA, 1% Triton X-100, 0.5 M guanidine-HCl, and 200 mMNaCl. DNase free RNase A was added at a concentration of 20 mg/liter andthe lysate was incubated at 37° C. for 30 minutes. Cellular debris wasremoved by centrifugation, and DNA was isolated using a QIAGEN Maxi 500column (QIAGEN Inc., Valencia, Calif.). The columns were equilibrated in10 ml of QBT, washed with 30 ml of QC, and eluted with 15 ml of QF (allbuffers from QIAGEN Inc., Valencia, Calif.). DNA was precipitated inisopropanol, washed in 70% ethanol, and recovered by centrifugation. TheDNA was resuspended in TE buffer.

Example 3 Cloning of an Aspergillus fumigatus Lipase 1 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify an Aspergillus fumigatus lipase 1 gene from the genomic DNAprepared in Example 2.

(SEQ ID NO: 17) Forward primer: 5′-GAGACGCATGCTTCACAAGTATAG-3′ (SEQ IDNO: 18) Reverse primer: 5′-GTCACCTCTAGTTAATTAATCAGATTATCTTGC-3′Bold letters represent coding sequence.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System (Roche Diagnostics, Mannheim, Germany). One μM ofeach of the primers above were used in a PCR reaction containing 20 ngof Aspergillus fumigatus genomic DNA, 1×PCR buffer (Roche Diagnostics,Mannheim, Germany) with 1.5 mM MgCl₂, 1 μl of a dATP, dTTP, dGTP, anddCTP mix (10 mM each), and 0.75 μl of DNA polymerase mix (3.5 U/μl;Roche Diagnostics, Mannheim, Germany) in a final volume of 50 μl. Toamplify the fragment, an Eppendorf Mastercycler Thermocycler (Hamburg,Germany) was programmed for 1 cycle at 94° C. for 2 minutes; 10 cycleseach at 94° C. for 15 seconds, 60° C. for 30 seconds, and 72° C. for1.25 minutes; 15 cycles each at 94° C. for 15 seconds, 60° C. for 30seconds, and 72° C. for 1.25 minutes plus a 5 second elongation at eachsuccessive cycle; 1 cycle at 72° C. for 7 minutes; and a 10° C. hold.

The reaction product was visualized on a 0.8% agarose gel using 44 mMTris Base, 44 mM boric acid, 0.5 mM EDTA (TBE) buffer and a 1.3 kbproduct band was purified using a QIAquick PCR Purification Kit (QIAGENInc., Valencia, Calif.) according to the manufacturer's instructions.

The PCR fragment and pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.)were ligated using conditions specified by the manufacturer to producepSMO224 (FIG. 1). Two μl of the reaction was used to transform E. coliTOP10 One Shot competent cells (Invitrogen, Carlsbad, Calif.) accordingto the manufacturer's instructions. A 2 μl volume of the ligationmixture was added to the E. coli cells and incubated on ice for 20minutes. Subsequently, the cells were heat shocked for 30 seconds at 42°C., and then placed on ice for 2 minutes. A 250 μl volume of SOC mediumwas added to the cells and the mixture was incubated for 1 hour at 37°C. and 250 rpm. After the incubation the colonies were spread on 2×YTplates supplemented with 100 μg of ampicillin per ml and incubated at37° C. overnight for selection of the plasmid. Six colonies that grew onthe plates were picked with a sterile toothpick and grown overnight at37° C., 250 rpm in a 15 ml Falcon tube containing 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. An E. coli transformantcontaining a plasmid designated pSMO224 was detected by restrictiondigestion and plasmid DNA was prepared using a BioRobot 9600 (QIAGENInc., Valencia, Calif.).

E. coli TOP10 One Shot cells containing pSMO224 were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30774, with a deposit date of Sep. 16, 2004.

Example 4 Characterization of the Aspergillus fumigatus Genomic SequenceEncoding Lipase 1

DNA sequencing of the Aspergillus fumigatus lipase 1 gene from pSMO224was performed with an Applied Biosystems Model 377 XL DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and primer walking strategy. Nucleotide sequence datawere scrutinized for quality and all sequences were compared to eachother with assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash.).

Gene models for the sequence were constructed based on the tfasty outputand alignment with a homologous lipase gene from Thermomyceslanuginosus. The nucleotide sequence (SEQ ID NO: 1) and deduced aminoacid sequence (SEQ ID NO: 2) are shown in FIG. 2. The genomic fragmentencodes a polypeptide of 396 amino acids, interrupted by 1 intron of 68bp. The % G+C content of the gene is 52.6% and the mature protein codingregion is 52.9%. Using the SignalP software program (Nielsen et al.,1997, Protein Engineering 10: 1-6), a signal peptide of 24 residues waspredicted. The predicted mature protein contains 373 amino acids with amolecular mass of 41.5 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus fumigatus lipase 1 geneshares 23% identity to the deduced amino acid sequence of a Thermomyceslanuginosus lipase gene (accession number 059952).

Example 5 Construction of pAlLo1 Expression Vector

Expression vector pAlLo1 was constructed by modifying pBANe6 (U.S. Pat.No. 6,461,837), which comprises a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase (NA2-tpi promoter), Aspergillus nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans acetamidase gene (amdS). Modification of pBANe6 was performedby first eliminating three Nco I restriction sites at positions 2051,2722, and 3397 bp from the amdS selection marker by site-directedmutagenesis. All changes were designed to be “silent” leaving the actualprotein sequence of the amdS gene product unchanged. Removal of thesethree sites was performed simultaneously with a GeneEditor Site-DirectedMutagenesis Kit (Promega, Madison, Wis.) according to the manufacturer'sinstructions using the following primers (underlined nucleotiderepresents the changed base):

(SEQ ID NO: 19) AMDS3NcoMut (2050): 5′-GTGCCCCATGATACGCCTCCGG-3′ (SEQ IDNO: 20) AMDS2NcoMut (2721): 5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′ (SEQ ID NO:21) AMDS1NcoMut (3396): 5′-GGAGGCCATGAAGTGGACCAACGG-3′

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QuickChange MutagenesisKit (Stratagene, La Jolla, Calif.), to eliminate the Nco I restrictionsite at the end of the AMG terminator at position 1643. The followingprimers (underlined nucleotide represents the changed base) were usedfor mutagenesis:

Upper Primer to mutagenize the AMG terminator sequence:

(SEQ ID NO: 22) 5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG- 3′Lower Primer to mutagenize the AMG terminator sequence:

(SEQ ID NO: 23) 5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG- 3′

The last step in the modification of pBANe6 was the addition of a newNco I restriction site at the beginning of the polylinker using aQuickChange Mutagenesis Kit and the following primers (underlinednucleotides represent the changed bases) to yield pAlLo1 (FIG. 3).

Upper Primer to mutagenize the NA2-tpi promoter:

(SEQ ID NO: 24) 5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′Lower Primer to mutagenize the NA2-tpi promoter:

(SEQ ID NO: 25) 5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

Example 6 Construction of pBM120a Expression Vector

Plasmid pBM120a was constructed to obtain a plasmid containing thedouble NA2 promoter (NA2-NA2-tpi) for driving gene expression inAspergillus species, and containing the ampicillin resistance gene forselection in E. coli.

Primers were designed to PCR amplify the double NA2 promoter frompJaL721 (WO 03/008575). Restriction enzyme sites Sal I and Nco I(underlined) were added for cloning the double promoter into theAspergillus expression plasmid pAlLo1.

(SEQ ID NO: 26) 5′-GTCGACATGGTGTTTTGATCATTTTA-3′ (SEQ ID NO: 27)5′-CCATGGCCAGTTGTGTATATAGAGGA-3′

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. The PCR amplification reaction mixture contained 1μl of 0.09 μg of pJaL721 per μl, 1 μl of each of the primers (50pmol/μl), 5 μl of 10×PCR buffer with 15 mM MgCl₂, 1 μl of a dATP, dTTP,dGTP, and dCTP mix (10 mM each), 37.25 μl of water, and 0.75 μl of DNApolymerase mix (3.5 U/μl). To amplify the fragment, an EppendorfMastercycler thermocycler was programmed for 1 cycle at 94° C. for 2minutes; 10 cycles each at 94° C. for 15 seconds, 55° C. for 30 seconds,and 72° C. for 1.25 minutes; 15 cycles each at 94° C. for 15 seconds,55° C. for 30 seconds, and 72° C. for 1.25 minutes plus a 5 secondelongation at each successive cycle; 1 cycle at 72° C. for 7 minutes;and a 10° C. hold. Ten microliters of this PCR reaction was mixed with 1μl of 10×DNA loading dye (25% glycerol, 10 mM Tris pH 7.0, 10 mM EDTA,0.025% bromophenol blue, 0.025% xylene cyanol) and run on a 1.0% (w/v)agarose gel using TBE buffer. The 1128 bp PCR product was observed withUV light on a Nucleotech gel visualization system (Nucleotech, SanMateo, Calif.). The PCR product was directly ligated into pPC2.1-TOPOaccording to the manufacturer's instructions. A 1 μl volume of fresh PCRproduct, 3 μl of double-distilled water, and 1 μl of the TOPO cloningvector were mixed with a pipette and incubated at room temperature for 5minutes.

After the incubation, 2 μl of the mixture was used to transform OneShotcompetent E. coli cells. A 2 μl volume of the ligation mixture was addedto the E. coli cells and incubated on ice for 5 minutes. Subsequently,the cells were heat shocked for 30 seconds at 42° C., and then placed onice for 2 minutes. A 250 μl volume of SOC medium was added to the cellsand the mixture was incubated for 1 hour at 37° C. and 250 rpm. Afterthe incubation the colonies were spread on 2×YT plates supplemented with100 μg of ampicillin per ml and incubated at 37° C. overnight forselection of the plasmid. Eight colonies that grew on the plates werepicked with a sterile toothpick and grown overnight at 37° C., 250 rpmin a 15 ml Falcon tube containing 3 ml of LB medium supplemented with100 μg of ampicillin per ml. The plasmids were isolated using a QIAGENBioRobot 9600.

Four μl volumes of the resulting plasmid minipreps were digested withEco RI. The digestion reactions were analyzed by agarose gelchromatography and UV analysis as previously described for the PCRreaction. Isolated plasmids containing an insert were sequenced using 1μl of plasmid template, 1.6 ng of M13 primer (forward or reverse) (MWGBiotech; High Point; NC), and water to 6 μl. DNA sequencing wasperformed with an Applied Biosystems Model 377 Sequencer XL (AppliedBiosystems, Inc., Foster City, Calif.) using dye-terminator chemistry.The resulting plasmid was designated pBM121b (FIG. 4).

A 5 μl volume of pBM121b was digested with Sal I and Nco I. Thedigestion reactions were analyzed by agarose gel electrophoresis asdescribed above, and ligated to the vector pAlLo1, which had beenpreviously digested with Sal I and Nco I. The resulting expressionplasmid was designated pBM120a (FIG. 5).

Example 7 Construction of an Aspergillus oryzae Expression VectorExpressing Aspergillus fumigatus Lipase 1 Gene

Two synthetic oligonucleotide primers shown below were designed to PCRamplify the Aspergillus fumigatus lipase 1 gene from the genomic DNAprepared in Example 2.

(SEQ ID NO: 28) Forward primer: 5′-TACACAACTGGCCATGCTTCACAAGTATAG-3′(SEQ ID NO: 29) Reverse primer: 5′-GTCACCTCTAGTTAATTAATCAGATTATCTTGC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. One μM of each of the primers above were used in aPCR reaction containing 20 ng of Aspergillus fumigatus genomic DNA,1×PCR buffer (Roche Diagnostics, Mannheim, Germany) with 1.5 mM MgCl₂, 1μl of a dATP, dTTP, dGTP, and dCTP mix (10 mM each), and 0.75 μl of DNApolymerase mix (3.5 U/μl; Roche Diagnostics, Mannheim, Germany) in afinal volume of 50 μl. To amplify the fragment, an EppendorfMastercycler thermocycler was programmed for 1 cycle at 94° C. for 2minutes; 10 cycles each at 94° C. for 15 seconds, 60° C. for 30 seconds,and 72° C. for 1.25 minutes; 15 cycles each at 94° C. for 15 seconds,60° C. for 30 seconds, and 72° C. for 1.25 minutes plus a 5 secondelongation at each successive cycle; 1 cycle at 72° C. for 7 minutes;and a 10° C. hold.

The reaction product was visualized on a 0.8% agarose gel using TBEbuffer and a 1.3 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions.

The 1.3 kb PCR fragment containing the Aspergillus fumigatus lipase 1gene was cloned into pBM120a using an InFusion Cloning Kit (BDBiosciences, Palo Alto, Calif.) where the vector was digested with Nco Iand Pac I. The digested vector was purified by gel electrophoresis usinga 0.7% agarose gel with TBE buffer, and the PCR fragment was extractedusing a QIAquick Gel Extraction Kit (QIAGEN Inc., Valencia, Calif.) andpurified using a QIAquick PCR Purification Kit. The gene fragment andthe digested vector were ligated together in a reaction resulting in theexpression plasmid pSMO218 (FIG. 6). The ligation reaction (50 μl) wascomposed of 1× InFusion Buffer (BD Biosciences, Palo Alto, Calif.),1×BSA (BD Biosciences, Palo Alto, Calif.), 1 μl of Infusion enzyme(diluted 1:10) (BD Biosciences, Palo Alto, Calif.), 100 ng of pBM120adigested with Nco I and Pac I, and 50 ng of the Aspergillus fumigatuslipase 1 gene purified PCR product. The reaction was incubated at roomtemperature for 30 minutes. Two μl of the reaction was used to transformE. coli SoloPack® Gold supercompetent cells (Stratagene, La Jolla,Calif.) according to the manufacturer's instructions. One μl ofβ-mercaptoethanol was added to the competent cells, and incubated on icefor 10 minutes. A 2 μl volume of the ligation mixture was then added tothe E. coli cells and incubated on ice for 30 minutes. Subsequently, thecells were heat shocked for 60 seconds at 54° C., and then placed on icefor 2 minutes. A 150 μl volume of NZY⁺ medium at 42° C. was added to thecells and the mixture was incubated for 1 hour at 37° C. and 250 rpm.After the incubation the colonies were spread on 2×YT platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.overnight for selection of the plasmid. Six colonies that grew on theplates were picked with a sterile toothpick and grown overnight at 37°C., 250 rpm in a 15 ml Falcon tube containing 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. An E. coli transformantcontaining the pSMO218 plasmid was detected by restriction digestion andplasmid DNA was prepared using a QIAGEN BioRobot 9600.

Example 8 Expression of the Aspergillus fumigatus Lipase 1 Gene inAspergillus oryzae BECh2

Aspergillus oryzae BECh2 (Δalp, Δamy, CPA-, KA-, Δnp1) protoplasts wereprepared according to the method of Christensen et al., 1988,Bio/Technology 6: 1419-1422. Five μg of pSMO218 was used to transformAspergillus oryzae BECh2.

The transformation of Aspergillus oryzae BECh2 with pSMO218 yielded 25transformants. The transformants were isolated to individual Coveplates. Confluent Cove plates of 25 transformants were washed with 4 mlof 0.01% Tween 20. Two hundred μl of spore suspension was inoculatedseparately into 25 ml of MY25 medium in 125 ml plastic shake flasks andincubated at 34° C., 250 rpm. Three and five days after incubation,culture supernatants were removed for lipase assay and SDS-PAGEanalysis.

Lipase activity was determined as follows: 200 μl of substrate (20 ml of100 mM MOPS pH 7.5, 4.95 ml DMSO, and 50 μl of p-nitrophenyl butyrate)was added to 20 μl of diluted enzyme sample. The samples were dilutedaccordingly in 100 mM MOPS pH 7.5, 4 mM CaCl₂, and 0.01% Triton X-100.The absorbance at 405 nm was obtained after 15 minutes of incubation atroom temperature (25° C.) in a 96-well microtiter plate using aSpectraMAX 250 microplate reader (Molecular Devices Corp., Sunnyvale,Calif.). LIPOLASE™ (Thermomyces lanuginosus lipase; Novozymes A/S,Bagsvaerd, Denmark)) can be used for generating a standard curve todetermine lipase units (LUs).

The lipase assay results indicated that at 3 days several of thetransformants produced lipase activity above that of the untransformedcontrol. SDS-PAGE analysis (BioRad Criterion 10-20% SDS-PAGE) of 10 μlof the supernatants showed a band at approximately 41 kDa.

Example 9 Determination of Substrate Specificity of RecombinantAspergillus fumigatus Lipase 1

The substrate specificity of Aspergillus fumigatus lipase 1 wasdetermined using a panel screen composed of 4-nitrophenol (PNP) lipasesubstrates.

A panel screen composed of a set of 12 assays utilizing various4-nitrophenol (PNP) lipase substrates was prepared as described in theTable 1.

TABLE 1 Panel screen of PNP substrates and buffer conditions 1 mM PNP-PNP tagged Shorthand 50 mM MOPS 50 mM CHES 50 mM MOPS 10 mM TritonConversion substrate designation pH 7.0 pH 9.5 pH 7.5 CaCl₂ X-100Factors 1 Palmitate 16:0 x x 1.2% 4.466 2 Palmitate 16:0 x x 1.2% 1.1 3Palmitate 16:0 x x 1.2% 2.037 4 Palmitate 16:0 x x 0.2% 1.0 5 Palmitate16:0 x x 0.2% 1.495 6 Decanoate 10:0 x x 1.2% 4.466 7 Decanoate 10:0 x x1.2% 1.1 8 Decanoate 10:0 x x 1.2% 2.037 9 Decanoate 10:0 x x 0.2% 1.010 Valerate  5:0 x x 0.40%  1.630 11 Valerate  5:0 x x   0% 1.370 12Butyrate  4:0 x x 0.40%  1.630

These assays were run in 384-well plates using 8 different dilutions ofeach sample (7 μl) to be evaluated and 80 μl of the substrates. Theassays were incubated for up to 24 hours at ambient temperature. Assayswere read at 405 nm at time points of approximately 1, 2, 3, 5, and 24hours. The results were calculated as OD/hour for each individual assay.In order to make an accurate evaluation of the amount of PNP released,it was necessary to mathematically normalize raw OD values by using aconversion factor. The conversion factors were values, determinedexperimentally, that were necessary to compensate for the fact that PNPhas lower OD readings at low pH and in the presences of detergent(Triton X100) than at pH 9.5. The factor normalizes the data to the ODreading that would have been obtained were it possible to quench thereactions to yield maximal OD for each condition while also stopping thereaction at that time point; i.e., PNP-fatty acid substrates are notstable at high pH, the tag comes off without lipase present at high pH,and the tagged substrate is particularly unstable above pH 9 and forshorter chain length fatty acid substrates.

In Table 1 the top two rows (1 and 2) were the assays used for the pHratio (9.5/7.0). Rows 3 and 10 were used for the “long chain (Slu)/shortchain (Lu); comparisons.

The results for the panel screen are shown in Table 2.

TABLE 2 Ratio of PNP-substrates according to Table 1, Example 9, where:P = Palmitate, D = Decanoate, V = Valerate and B = Butyrate Longchain/Short chain at pH 7.5 P/V D/V P/B D/B ALF004 1.888 0.887 2.5561.201 LIPOLASE ™ 1.213 2.398 2.220 4.387 pH Ratios; (−) = 0.2% Tritondata 9.5P/7.0P 9.5D/7.0D 9.5P−/7.5P− ALF004 0.045 No Data Not TestedLIPOLASE ™ 1.527 1.881 1.452 No or Low Triton (−) compared to maximumTriton (+) V7.5−/+ D9.5+/− P7.5+/− P9.5+/− ALF004 Not Tested Not TestedNot Tested Not Tested LIPOLASE ™ 8.651 6.378 9.206 9.502 No Data = notenough activity to give sufficient signal in one of the assays in thistest ALF004 (Aspergillus oryzae BeCH2 expressing Aspergillus fumigatuslipase 1) test data consists of the average of up to 2 independentscreening assay events; LIPOLASE ™ test data consists of the average ofup to 17 independent screening assay events.

In comparing the panel screen results of the Thermomyces lanuginosuslipase (LIPOLASE™) and the ALF004 the following observations were made:

1. The ratio of activities on PNP-palmitate at pH 9.5 versus pH 7 is40-fold lower for ALF004 versus LIPOLASE™ suggesting that theAspergillus fumigatus lipase 1 has much lower activity at pH 9.5 versusLipolase™ or it has a much higher activity at pH 7.0 than LIPOLASE™ or acombination of these two.

2. The ratios of P/V, D/V, and D/B are also quite different for ALF004versus LIPOLASE™ suggesting there is some acyl change length specificitydifferences between the two lipases.

Example 10 Cloning of an Aspergillus fumigatus Lipase 2 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify an Aspergillus fumigatus lipase 2 gene from the genomic DNAprepared in Example 2.

(SEQ ID NO: 30) Forward primer: 5′-GAGACACATGTTTCACCCAG-3′ (SEQ ID NO:31) Reverse primer: 5′-GTCACCTCTAGTTAATCAGTTAGTTGAGC-3′Bold letters represent coding sequence.

The fragment was amplified by PCR using the Expand High Fidelity PCRSystem as described in Example 3. The reaction product was visualized ona 0.7% agarose gel using TBE buffer and a 1.2 kb product band waspurified using a QIAquick PCR Purification Kit according to themanufacturer's instructions. The PCR product was then cloned intopCR2.1-TOPO according to manufacturer's instructions to produce pSMO223(FIG. 7). Two μl of the reaction was used to transform E. coli TOP10 OneShot competent cells according to the manufacturer's instructions. A 2μl volume of the ligation mixture was added to the E. coli cells andincubated on ice for 20 minutes. Subsequently, the cells were heatshocked for 30 seconds at 42° C., and then placed on ice for 2 minutes.A 250 μl volume of SOC medium was added to the cells and the mixture wasincubated for 1 hour at 37° C. and 250 rpm. After the incubation thecolonies were spread on 2×YT plates supplemented with 100 μg ofampicillin per ml and incubated at 37° C. overnight for selection of theplasmid. Six colonies that grew on the plates were picked with a steriletoothpick and grown overnight at 37° C., 250 rpm in a 15 ml Falcon tubecontaining 3 ml of LB medium supplemented with 100 μg of ampicillin perml. An E. coli transformant containing pSMO223 was detected byrestriction digestion and plasmid DNA was prepared using a QIAGENBioRobot 9600.

E. coli TOP10 One Shot cells containing pSMO223 were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30773, with a deposit date of Sep. 16, 2004.

Example 11 Characterization of the Aspergillus fumigatus GenomicSequence Encoding Lipase 2

DNA sequencing of the Aspergillus fumigatus lipase 2 gene from pSMO223was performed with an Applied Biosystems Model 377 XL DNA Sequencerusing dye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand sequences were compared to each other with assistance of PHRED/PHRAPsoftware (University of Washington, Seattle, Wash.).

Gene models for the sequence were constructed based on the tfasty outputand alignment with a homologous lipase gene from Thermomyceslanuginosus. The nucleotide sequence (SEQ ID NO: 3) and deduced aminoacid sequence (SEQ ID NO: 4) are shown in FIG. 8. The genomic fragmentencodes a polypeptide of 283 amino acids, interrupted by 2 introns of 45and 50 bp. The % G+C content of the gene is 47.1% and the mature proteincoding region is 47.2%. Using the SignalP software program (Nielsen etal., 1997, supra), a signal peptide of 24 residues was predicted. Thepredicted mature protein contains 259 amino acids with a molecular massof 28.9 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus fumigatus lipase 2 geneshares 35% identity to the deduced amino acid sequence of a Thermomyceslanuginosus lipase gene (accession number 059952).

Example 12 Identification of Lipase Genes in the Partial GenomicSequence of Magnaporthe grisea

A tfasty search (Pearson, W. R., 1999, supra) of the Magnaporthe griseapartial genome sequence (Broad Institue MIT, Boston, Mass.) was carriedout using as query a lipase sequence from Thermomyces lanuginosus(Accession No. 059952). Several genes were identified as putativelipases based upon an E value of less than 0.001 in the tfasty output.Three genomic regions of approximately 930 bp, 750 bp, and 720 bp withgreater than 30% identity to the query sequence at the amino acid levelwere chosen for further study. Gene models for the putative lipase geneswere predicted based on homology to the Thermomyces lanuginosus lipaseas well as conserved sequences present at the 5′ and 3′ ends of fungalintrons.

Example 13 Magnaporthe grisea Genomic DNA Extraction

Four hundred μl of Magnaporthe grisea (FGSC 8958, Fungal Genetics StockCenter) spores were grown in 50 ml of CM medium in a baffled shake flaskat 25° C. and 250 rpm for 4 days. Genomic DNA was then extracted fromthe mycelia using a DNeasy Plant Mini Kit (QIAGEN, Valencia, Calif.)according to manufacturer's instructions.

Example 14 Cloning of Magnaporthe grisea Lipase 1 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe genomic sequence outside of the predicted start and stop codons ofthe open reading frame to PCR amplify a Magnaporthe grisea lipase 1 genefrom the genomic DNA prepared in Example 13.

Forward primer: 5′-CCTTGCCCACGCCTTTGGTTC-3′(SEQ ID NO: 32) Reverseprimer: 5′-CTCATAGCAGCAGGCGAAGCC-3′(SEQ ID NO: 33)Both primers represent sequence outside of coding sequence.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 300 ng of Magnaporthe grisea genomic DNA, 1× Herculasereaction buffer (Stratagene, La Jolla, Calif.), 1 μl of a dATP, dTTP,dGTP, and dCTP mix (10 mM each), and 2.5 units of Herculase Hotstart DNApolymerase (Stratagene, La Jolla, Calif.) in a final volume of 50 μl.The amplification was conducted in an Eppendorf Mastercyclerthermocycler programmed for one cycle at 98° C. for 2 minutes; 10 cycleseach at 98° C. for 15 seconds, 62° C. for 30 seconds, and 72° C. for 1minute and 20 seconds; 15 cycles each at 98° C. for 15 seconds, 62° C.for 30 seconds, and 72° C. for 1 minutes and 20 seconds plus a 5 secondelongation at each successive cycle; and 1 cycle at 72° C. for 7minutes. The heat block then went to a 10° C. soak cycle.

The reaction products were run on a 1.0% agarose gel using 40 mM Trisbase-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a 1.2 kbproduct band was detected. PCR products were purified using a QIAquickPCR Purification Kit according to the manufacturer's instructions.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Magnaporthe grisea gene encoding a lipase 1 gene from the PCRproduct described above.

(SEQ ID NO: 34) Forward primer:5′-ACACAACTGGCCATGAAGGTCTCGTTCGTGTCATCG-3′ (SEQ ID NO: 35) Reverseprimer: 5′-AGTCACCTCTAGTTATCAGTAGCAAGCGCTAATGG-3′Bold letters represent coding sequence. The remaining sequence ishomologous to insertion sites of pBM120a.

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 2 μl of the 1.2 kb PCR product described above, 1× Herculasereaction buffer, 1 μl of a dATP, dTTP, dGTP, and dCTP mix (10 mM each),2.5 units of Herculase Hotstart DNA polymerase in a final volume of 50μl. The amplification was conducted in an Eppendorf Mastercyclerthermocycler programmed for 1 cycle at 98° C. for 2 minutes; 10 cycleseach at 98° C. for 15 seconds, 62° C. for 30 seconds, and 72° C. for 1minute and 20 seconds; 15 cycles each at 98° C. for 15 seconds, 62° C.for 30 seconds, and 72° C. for 1 minutes and 20 seconds plus a 5 secondelongation at each successive cycle; and 1 cycle at 72° C. for 7minutes. The heat block then went to a 10° C. soak cycle.

The reaction products were run on a 1.0% agarose gel using TAE bufferwhere a 1.1 kb product band was detected. The 1.1 kb PCR product waspurified using a QIAquick PCR Purification Kit according to themanufacturer's instructions.

The 1.1 kb fragment was cloned into the pCR2.1-TOPO vector. The genefragment was purified using a QIAquick PCR Purification Kit according tothe manufacturer's instructions. The fragment and pCR2.1-TOPO vectorwere ligated by using conditions specified by the manufacturer resultingin plasmid pHyGe026 (FIG. 9). Two μl of the reaction was used totransform E. coli One Shot competent cell. An E. coli transformantcontaining the plasmid pHyGe026 was detected by restriction digestionand plasmid DNA was prepared using a BioRobot 9600.

E. coli TOP10 One Shot cells containing pHyGe026 were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30772, with a deposit date of Sep. 13, 2004.

Example 15 Characterization of the Magnaporthe grisea Genomic SequenceEncoding a Lipase 1 Gene

DNA sequencing of the Magnoporthe grisea lipase 1 gene from pHyGe026 wasperformed with an Applied Biosystems Model 377 XL DNA Sequencer usingdye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand sequences were compared to each other with assistance of PHRED/PHRAPsoftware (University of Washington, Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to aThermomyces lanuginosa lipase (Accession No. 059952) as well asconserved sequences present at the 5′ and 3′ ends of fungal introns. Thenucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQID NO: 6) are shown in FIGS. 10A and 10B. The genomic fragment encodes apolypeptide of 318 amino acids, interrupted by 2 introns of 54 and 77bp. The % G+C content of the gene is 60.9% and the mature polypeptidecoding sequence (nucleotides 58 to 1088 of SEQ ID NO: 6) is 60.5%. Usingthe SignalP software program (Nielsen et al., 1997, supra), a signalpeptide of 19 residues was predicted. The predicted mature proteincontains 299 amino acids with a molecular mass of 34 kDa.

A comparative alignment of lipase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Magnaporthe lipase 1 gene shares31.3% identity to the deduced amino acid sequence of a Thermomyceslanuginosus lipase gene (Accession No. 059952).

Example 16 Construction of an Aspergillus oryzae Expression Vector forthe Magnaporthe grisea Lipase 1 Gene

The PCR fragment containing the Magnaporthe grisea lipase 1 gene wascloned into pBM120a using an InFusion Cloning Kit. The vector wasdigested with Nco I and Pac I. The digested vector was purified by gelelectrophoresis and extracted using a QIAquick Gel Extraction Kit. Thegene fragment and digested vector were ligated together in a reactionresulting in the expression plasmid pHyGe010 (FIG. 11) in whichtranscription of the lipase gene was under the control of the tandemNA2-tpi promoter. The ligation reaction (20 μl) was composed of 1×InFusion Buffer, 1×BSA, 1 μl of Infusion enzyme (diluted 1:10), 50 ng ofpBM120a digested with Nco I and Pac I, and 30 ng of the Magnaporthegrisea lipase purified PCR product. The reaction was incubated at roomtemperature for 30 minutes. Two μl of the reaction was used to transformE. coli SoloPack® Gold supercompetent cells according to manufacturer'sinstructions. An E. coli transformant containing pHyGe010 was detectedby estriction digestion and plasmid DNA was prepared using a QIAGENBioRobot 9600.

Example 17 Expression of the Magnaporthe grisea Lipase 1 Gene inAspergillus oryzae BECh2

Aspergillus oryzae BECh2 protoplasts were prepared according to themethod of Christensen et al., 1988, supra. Sixty μg of pHyGe010 was usedto transform Aspergillus oryzae BECh2.

The transformation of Aspergillus oryzae BECh2 with pHyGe010 yielded 14transformants. Each transformant was transferred to individual Coveplates. Confluent Cove plates of the 14 transformants were washed with 4ml of 0.01% Tween 20. Two hundred μl of spore suspension was inoculatedseparately into 25 ml of MY25 medium in 125 ml plastic shake flasks andincubated at 34° C., 250 rpm. Three, four, and five days afterincubation, culture supernatants were removed for lipase assay andSDS-PAGE analysis.

Lipase activity was determined as described in Example 8.

The lipase assay results indicated that at 3, 4, and 5 days, severaltransformants produced lipase activity above that of the untransformedcontrol.

SDS-PAGE (BioRad Criterion 8-16% SDS-PAGE) analysis of 25 μl of thesupernatants showed a major band at approximately 37 kDa.

Example 18 Determination of Substrate Specificity of RecombinantMagnaporthe grisea Lipase 1

The substrate specificity of Magnaporthe grisea lipase 1 was determinedusing a panel screen composed of 4-nitrophenol (PNP) lipase substratesas described in Example 9.

The results for the panel screen are shown in Table 3.

TABLE 3 Ratio of PNP-substrates according to Table 1, Example 9, where:P = Palmitate, D = Decanoate, V = Valerate and B = Butyrate Longchain/Short chain at pH 7.5 P/V D/V P/B D/B Hyge027 1.142 0.747 1.6571.103 LIPOLASE ™ 1.213 2.398 2.220 4.387 pH Ratios; (−) = 0.2% Tritondata 9.5P/7.0P 9.5D/7.0D 9.5P−/7.5P− Hyge027 No Data No Data No DataLIPOLASE ™ 1.527 1.881 1.452 No or Low Triton (−) compared to maximumTriton (+) V7.5−/+ D9.5+/− P7.5+/− P9.5+/− Hyge027 1.101 No Data 5.123No Data LIPOLASE ™ 8.651 6.378 9.206 9.502 No Data = not enough activityto give sufficient signal in one of the assays in this test Hyge027(Aspergillus oryzae BECh2 expressing Magnaporthe grisea lipase 1) testdata consists of the average of up to 4 independent screening assayevents; LIPOLASE ™ (Thermomyces lanuginosus lipase) test data consistsof the average of up to 17 independent screening assay events.

In comparing the panel screen results of the Thermomyces lanuginosuslipase (LIPOLASE™) and Hyge027, the following observations were made:

-   -   (1) There were significant differences in the ratios of        activities on valerate or palmitate at pH 7.5 (V7.5 −/+ and P7.5        +/−) in the presence or absence of triton for Hyge027 versus        LIPOLASE™.

(2) The ratios of activities on decanoate versus valerate (D/V),palmitate versus butyrate (P/B), and decanoate versus butyrate (D/B)were significantly different for Hyge027 versus LIPOLASE™ suggestingthere is some acyl change length specificity differences between the twolipases.

Example 19 Cloning of Magnaporthe grisea Lipase 2 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify a Magnaporthe grisea lipase 2 gene from the genomic DNA preparedin Example 13.

(SEQ ID NO: 36) Forward primer: 5′-CCATGGCCATGATGAGGTTCCCCAGCGTGCTCA-3′(SEQ ID NO: 37) Reverse primer: 5′-TTTAATTAAGCCACGGTCTTGTTGGCTTC-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the enzyme restriction insertion sites of pBM120a.

The gene was amplified by PCR using the Herculase™ Hotstart DNApolymerase (Stratagene, La Jolla, Calif.). Fifty ng of each of theprimers above were used in a PCR reaction containing 300 ng ofMagnaporthe grisea genomic DNA. The PCR amplification reaction mixturealso contained 1× Herculase PCR buffer, 1 μl of a dATP, dTTP, dGTP, anddCTP mix (10 mM each), and 2.5 units Herculase™ DNA polymerase mix in afinal volume of 50 μl. To amplify the fragment, a Peltien thermal cycler(MJ Research Inc., Watertown, Mass.) was programmed for 1 cycle at 94°C. for 2 minutes; 30 cycles each at 94° C. for 1 minute, 50° C. for 1minute, 72° C. 1 minute; 1 cycle at 72° C. for 10 minutes; and a 4° C.hold.

The PCR products were size fractionated on a 1% agarose gel using TAEbuffer and a 1 kb product band was purified using a QIAquick GelPurification Kit according to the manufacturer's instructions. The PCRproduct was then cloned into pCR2.1-TOPO according to manufacturer'sinstructions resulting in plasmid pCrAm138 (FIG. 12). Two μl of theligation reaction was used to transform E. coli TOP10 One Shot competentcells. An E. coli transformant containing the plasmid pCrAm138 wasdetected by restriction digestion and plasmid DNA was prepared using aQIAGEN BioRobot 9600.

E. coli TOP10 One Shot cells containing pCrAm138 were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30781, with a deposit date of Oct. 12, 2004.

Example 20 Characterization of the Magnaportha grisea Genomic SequenceEncoding Lipase 2

DNA sequencing of the Margnoportha grisea lipase 2 gene contained inpCrAm138 was performed with a Perkin-Elmer Applied Biosystems Model 377XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke etal., 1992, supra) and primer walking strategy. Nucleotide sequence datawere scrutinized for quality and all sequences were compared to eachother with assistance of PHRED/PHRAP software (University of Washington,Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to aThermomyces lanuginosus lipase (Accession No. 059952) as well asconserved sequences present at the 5′ and 3′ ends of fungal introns. Thenucleotide sequence (SEQ ID NO: 7) and deduced amino acid sequence (SEQID NO: 8) are shown in FIG. 13. The genomic fragment encodes apolypeptide of 348 amino acids with a molecular weight of 37.7 kDa. The% G+C content of the gene is 64% and the mature polypeptide codingsequence (nucleotides 55 to 1044 of SEQ ID NO: 7) is 64%. Using theSignalP software program (Nielsen et al., 1997, supra), a signal peptideof 18 residues was predicted. The predicted mature protein contains 330amino acids with a molecular mass of 35.9 kDa.

A comparative alignment of lipase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Magnaporthe lipase 2 gene shares37.8% identity to the deduced amino acid sequence of a Thermomyceslanuginosus lipase gene (Accession No. 059952).

Example 21 Cloning of Magnaporthe grisea Lipase 3 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify a Magnaporthe grisea lipase 3 gene from the genomic DNAdescribed in Example 13.

(SEQ ID NO: 38) Forward primer:5′-ACACAACTGGCCATGTTGTGGCGTCGGGCGGGTGGCCTCT-3′ (SEQ ID NO: 39) Reverseprimer: 5′-AGTCACCTCTAGTTAATTAATTAGAGCTCATCCTGGCCAGGAGCCA C-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. Fifty picomoles of each of the primers above wereused in a PCR reaction containing 300 ng of Magnaporthe grisea genomicDNA. The PCR amplification reaction mixture also contained 1×PCR bufferwith 1.5 mM MgCl₂, 1 μl of a dATP, dTTP, dGTP, and dCTP mix (10 mMeach), and 0.75 μl of DNA polymerase mix (3.5 U/μl) in a final volume of501 μl. An Eppendorf Mastercycler thermocycler was used to amplify thefragment programmed for 1 cycle at 94° C. for 2 minutes; 10 cycles eachat 94° C. for 15 seconds, 62° C. for 30 seconds, and 72° C. for 2minutes; 15 cycles each at 94° C. for 15 seconds, 62° C. for 30 seconds,and 72° C. for 2 minutes plus a 5 second elongation at each successivecycle; 1 cycle at 72° C. for 7 minutes; and a 10° C. hold.

The 1.2 kb reaction product was visualized on a 1.0% agarose gel usingTBE buffer. Four microliters of the product was then cloned intopCR2.1-TOPO according to manufacturer's instructions to produce pBM135a.Two μl of the reaction was used to transform E. coli TOP10 One Shotcompetent cells. An E. coli transformant containing pBM135a was detectedby restriction digestion and plasmid DNA was prepared using a QIAGENBioRobot 9600.

DNA sequencing of the Magnaporthe grisea lipase 3 gene from pBM135a wasperformed with a Perkin-Elmer Applied Biosystems Model 377 XL AutomatedDNA Sequencer using dye-terminator chemistry (Giesecke et al., 1992,supra) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were analyzed with assistanceof ContigExpress software (Informax, Inc., Bethesda, Md.). Sequencingresults indicated the absence of the ATG start codon in pBM135a. Asecond forward primer, with sequence identical to oligo number 998205described above was ordered. The new oligo numbered 998524 was used in aPCR reaction using pBM135a as DNA template to re-amplify the gene ofinterest.

Using the Expand High Fidelity PCR System, 50 picomoles of primers998524 and 998232 were used in a PCR reaction containing 1 μl of a 1:10dilution pBM135a mini DNA. The PCR amplification reaction mixture alsocontained 1×PCR buffer with 1.5 mM MgCl₂, 1 μl of a dATP, dTTP, dGTP,and dCTP mix (10 mM each), and 0.75 μl of DNA polymerase mix (3.5 U/μl)in a final volume of 50 μl. An Eppendorf Mastercycler thermocycler wasused to amplify the fragment programmed for 1 cycle at 94° C. for 2minutes; 10 cycles each at 94° C. for 15 seconds, 62° C. for 30 seconds,and 72° C. for 1 minute, 15 seconds; 15 cycles each at 94° C. for 15seconds, 62° C. for 30 seconds, and 72° C. for 1 minute 15 seconds, plusa 5 second elongation at each successive cycle; 1 cycle at 72° C. for 7minutes; and a 10° C. hold.

The 1.2 kb reaction product was visualized on a 1.0% agarose gel usingTBE buffer. The 1.2 kb fragment was excised from the gel and purifiedusing a Qiagen Gel Extraction Kit. Two microliters of the product wasthen cloned into pCR2.1-TOPO according to manufacturer's instructions toproduce pBM135g (FIG. 14). Two μl of the reaction was used to transformE. coli TOP10 One Shot competent cells. An E. coli transformantcontaining pBM135g was detected by restriction digestion and plasmid DNAwas prepared using a QIAGEN BioRobot 9600.

E. coli TOP10 One Shot cells containing pBM135g were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30779, with a deposit date of Oct. 12, 2004.

Example 22 Characterization of the Magnaporthe grisea Genomic SequenceEncoding Lipase 3

DNA sequencing of the Magnaporthe grisea lipase 3 gene from plasmid,pBM135g, was performed with a Perkin-Elmer Applied Biosystems Model 377XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke etal., 1992, supra) and primer walking strategy. Nucleotide sequence datawere scrutinized for quality and all sequences were analyzed withassistance of ContigExpress software.

Gene models for the lipase gene were predicted based on homology to aThermomyces lanuginosus lipase (Accession No. 059952) as well asconserved sequences present at the 5′ and 3′ ends of fungal introns. Thegenomic coding sequence (SEQ ID NO: 9) and deduced amino acid sequence(SEQ ID NO: 10) for pBM135g and is shown in FIG. 15. The genomicfragment encodes a polypeptide of 393 amino acids. The % G+C content ofthe gene is 58.71% and the mature polypeptide coding sequence(nucleotides 73 to 1179 of SEQ ID NO: 9) is 58.4%. Using the SignalPsoftware program (Nielsen et al., 1997, supra), a signal peptide of 24residues was predicted. The predicted mature protein contains 369 aminoacids with a molecular mass of 40.8 kDa.

A comparative alignment of lipase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Magnaporthe grisea lipase 3 geneshares 20.5% identity to the deduced amino acid sequence of theThermomyces lanuginosus lipase gene (Accession No. 059952).

Example 23 Identification of Lipase Genes in the Genomic Sequence ofAspergillus nidulans

A tfasty search (Pearson, W. R., 1999, supra) of the Aspergillusnidulans partial genome sequence (The Institute for Genomic Research,Rockville, Md.) was carried out using as query a lipase protein sequencefrom Thermomyces lanuginosus (Accession No. 059952). Several genes wereidentified as putative homologs based upon an E value of less than 0.001in the tfasty output. Three genomic regions of approximately 780 bp, 600bp, and 960 bp with 45.8, 34, and 33% identity to the query sequence atthe amino acid level were identified. Gene models for the putativelipase genes were predicted based on homology to the Thermomyceslanuginosus lipase as well as conserved sequences present at the 5′ and3′ ends of fungal introns.

Example 24 Aspergillus nidulans Genomic DNA Extraction

Four hundred μl of Aspergillus nidulans A1000 (Fungal Genetics StockCenter, Kansas City, Mo.) spores were grown in 50 ml of YP medium in abaffled shake flask at 37° C. and 250 rpm for 24 hours. Genomic DNA wasthen extracted from the mycelia using a DNeasy Plant Mini Kit accordingto manufacturer's instructions.

Example 25 Cloning of the Aspergillus nidulans Lipase 1 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify an Aspergillus nidulans gene encoding a lipase from the genomicDNA prepared in Example 24.

(SEQ ID NO: 40) Forward primer:5′-ACACAACTGGCCATGATCCGTTTGGGGTATTCTGCC-3′ (SEQ ID NO: 41) Reverseprimer: 5′-AGTCACCTCTAGTTAATTAATTACTGGCAGGCAGTGATAT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a (see Example 6).

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. One μM of each of the primers above were used in aPCR reaction containing 20 ng of Aspergillus nidulans genomic DNA, 1×PCRbuffer with 1.5 mM MgCl₂, 1 μl of a dATP, dTTP, dGTP, and dCTP mix (10mM each), and 0.75 μl of DNA polymerase mix (3.5 U/μl) in a final volumeof 50 μl. To amplify the fragment, an Eppendorf Mastercyclerthermocycler was programmed for 1 cycle at 94° C. for 2 minutes; 10cycles each at 94° C. for 15 seconds, 59.5° C. for 30 seconds, and 72°C. for 1.25 minutes; 15 cycles each at 94° C. for 15 seconds, 59.5° C.for 30 seconds, and 72° C. for 1.25 minutes plus a 5 second elongationat each successive cycle; 1 cycle at 72° C. for 7 minutes; and a 10° C.hold.

The reaction product was visualized on a 0.7% agarose gel using TBEbuffer and the 1.1 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions. The PCRfragment and pCR2.1-TOPO were ligated using conditions specified by themanufacturer resulting in plasmid pJLinl71 (FIG. 16).

Two μl of the reaction was used to transform E. coli TOP10 One Shotcompetent cells according to the manufacturer's instructions. A 2 μlvolume of the ligation mixture was added to the E. coli cells andincubated on ice for 20 minutes. Subsequently, the cells were heatshocked for 30 seconds at 42° C., and then placed on ice for 2 minutes.A 250 μl volume of SOC medium was added to the cells and the mixture wasincubated for 1 hour at 37° C. and 250 rpm. After the incubation thecolonies were spread on 2×YT plates supplemented with 100 μg ofampicillin per ml and incubated at 37° C. overnight for selection of theplasmid. Twelve colonies that grew on the plates were picked with asterile toothpick and grown overnight at 37° C., 250 rpm in a 15 mlFalcon tube containing 3 ml of LB medium supplemented with 100 μg ofampicillin per ml. An E. coli transformant containing the pJLin171 wasdetected by restriction digestion and plasmid DNA was prepared using aBioRobot 9600.

E. coli TOP 10 One Shot cells containing pJLin171 were deposited withthe Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30755, with a deposit date of Jul. 21, 2004.

Example 26 Characterization of the Aspergillus nidulans Genomic SequenceEncoding Lipase 1

DNA sequencing of the Aspergillus nidulans lipase 1 gene from pJLin171was performed with an Applied Biosystems Model 377 XL DNA Sequencerusing dye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand all sequences were compared to each other with assistance ofPHRED/PHRAP software (University of Washington, Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to theThermomyces lanuginosus lipase as well as conserved sequences present atthe 5′ and 3′ ends of fungal introns. The nucleotide sequence (SEQ IDNO: 11) and deduced amino acid sequence (SEQ ID NO: 12) are shown inFIG. 17. The genomic fragment encodes a polypeptide of 294 amino acids,interrupted by 3 introns of 47 bp, 59 bp, and 50 bp. The % G+C contentof the gene is 54.5% and of the mature protein coding region(nucleotides 58 to 1038 of SEQ ID NO: 11) is 54.5%. Using the SignalPsoftware program (Nielsen et al., 1997, supra), a signal peptide of 19residues was predicted. The predicted mature protein contains 275 aminoacids with a molecular mass of 29.4 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus nidulans lipase 1 geneshares 48% identity to the deduced amino acid sequence of a Thermomyceslanuginosus lipase gene (accession number 059952).

Example 27 Construction of an Aspergillus oryzae Expression VectorExpressing Aspergillus nidulans Lipase 1 Gene

The 1.1 kb PCR fragment (Example 25) containing the Aspergillus nidulanslipase 1 gene was cloned into pBM120a using an InFusion Cloning Kitwhere the vector was digested with Nco I and Pac I. The digested vectorwas purified by gel electrophoresis using a 0.7% agarose gel with TBEbuffer, and the PCR fragment was extracted using a QIAquick GelExtraction Kit and purified using a QIAquick PCR Purification Kit. Thegene fragment and the digested vector were ligated together in areaction resulting in the expression plasmid pJLin167 (FIG. 18). Theligation reaction (50 μl) was composed of 1× InFusion Buffer, 1×BSA, 1μl of Infusion enzyme (diluted 1:10), 100 ng of pBM120a digested withNco I and Pac I, and 50 ng of the Aspergillus nidulans lipase 1 genepurified PCR product. The reaction was incubated at room temperature for30 minutes. Two μl of the reaction was used to transform E. coliSoloPack® Gold supercompetent cells according to the manufacturer'sinstructions. One μl of β-mercaptoethanol was added to competent cells,and incubated on ice for 10 minutes. A 2 μl volume of the ligationmixture was then added to the E. coli cells and incubated on ice for 30minutes. Subsequently, the cells were heat shocked for 60 seconds at 54°C., and then placed on ice for 2 minutes. A 150 μl volume of NZY⁺ mediumat 42° C. was added to the cells and the mixture was incubated for 1hour at 37° C. and 250 rpm. After the incubation the colonies werespread on 2×YT plates supplemented with 100 μg of ampicillin per ml andincubated at 37° C. overnight for selection of the plasmid. Twelvecolonies that grew on the plates were picked with a sterile toothpickand grown overnight at 37° C., 250 rpm in a 15 ml Falcon tube containing3 ml of LB medium supplemented with 100 μg of ampicillin per ml. An E.coli transformant containing the pJLin167 plasmid was detected byrestriction digestion and plasmid DNA was prepared using a QIAGENBioRobot 9600.

Example 28 Expression of the Aspergillus nidulans Lipase 1 Gene inAspergillus oryzae BECh2

Aspergillus oryzae BECh2 (Δalp, Δamy, CPA-, KA-, Δnp1) protoplasts wereprepared according to the method of Christensen et al., 1988,Bio/Technology 6: 1419-1422. Five μg of pJLin167 was used to transformAspergillus oryzae BECh2.

The transformation of Aspergillus oryzae BECh2 with pJLin167 yielded 34transformants. The transformants were isolated to individual Coveplates. Confluent Cove plates of 28 transformants were washed with 4 mlof 0.01% Tween 20. Two hundred μl of spore suspension was inoculatedseparately into 25 ml of MY25 medium in 125 ml plastic shake flasks andincubated at 34° C., 250 rpm. Three and five days after incubation,culture supernatants were removed for lipase assay and SDS-PAGEanalysis.

Lipase activity was determined as described in Example 8. The lipaseassay results indicated that at both 3 and 5 days, 27 of the 28transformants produced lipase activity well above that of theuntransformed control.

SDS-PAGE (BioRad Criterion 10-20% SDS-PAGE) analysis of 10 μl of thesupernatants showed a major band at approximately 30 kDa.

Example 29 Purification and Characterization of Recombinant Aspergillusnidulans Lipase 1

One of the Aspergillus oryzae transformants producing the highest yieldof Aspergillus nidulans lipase 1 was grown in 500 ml of MY25 medium for4 days at 30° C., 250 rpm for purification. Supernatant was sterilefiltered under pressure using SEITZ-EKS filters (PALL Corporation,Waldstetten, Germany). The sterile filtered supernatant was thenadjusted to pH 9 and sodium chloride was added to a final concentrationof 2 M.

Decylamine agarose was custom made by UpFront Chromatography A/S, LersøParkalle Denmark. The decylamine agarose matrix was packed into a 50 mlcolumn and then washed and equilibrated with 50 mM borate pH 9 buffercontaining 2 M sodium chloride. Filtered fermentation supernatant wasthen applied to the column using an Akta explorer system. Unboundmaterial was washed with 50 mM borate pH 9 buffer containing 1 M sodiumchloride. The bound proteins were eluted with 50 mM borate pH 9 buffercontaining 30% 2-propanol as an eluent.

Fractions of 10 ml were collected and analyzed for lipase activityaccording to the assay described by Svendsen et al., in Methods inEnzymology, Lipases Part a Biotechnology Vol. 284 pages 317-340 Editedby Byron Rubin and Edward A. Dennis, Academic Press, 1997, New York.Fractions containing lipase activity were then pooled and diluted toadjust the ionic strength below 4 mSi.

A 50 ml FFQ Sepharose (Pharmacia Amersham, Uppsala, Sweden) column waswashed and equilibrated with 50 mM borate pH 9 buffer. The decylamineagarose pool containing activity was then applied onto the column andwashed with the same buffer to remove unbound material. Bound proteinswere then eluted using linear salt gradient using 50 mM borate pH 9buffer containing 0.5 M sodium chloride.

Fractions of 10 ml were collected and analyzed for activity as describedabove and fractions were also analyzed by SDS-PAGE for purity. Bestfractions contain highest lipase activity and best purity judged bySDS-PAGE was pooled. SDS-PAGE showed a pure protein band with molecularweight between 33 to 35 kDa protein which is usually seen due toglycosylation.

Substrate specificity of the Aspergillus nidulans lipase 1 was evaluatedat pH 7 according to WO 2005/040410. The results showed that theAspergillus nidulans lipase 1 efficiently degrades phospholipids such aslecithin (and alkylated phosphatidylethanolamins) and well astriliolein.

Example 30 Determination of Thermostability of Recombinant Aspergillusnidulans Lipase 1

The thermostability of purified recombinant Aspergillus nidulans lipase1 (Example 29) was determined by Differential Scanning Calorimetry(DSC). The thermal denaturation temperature, Td, was taken as the top ofthe denaturation peak (major endothermic peak) in thermograms (Cp vs. T)obtained after heating of enzyme solutions at a constant programmedheating rate. Cp refers to heat capacity (at constant pressure). Trefers to temperature.

A VP-DSC Differential Scanning Calorimeter (MicroCal Inc., Northampton,Mass.) was used for the thermostability determination according to themanufacturer's instructions. Sample enzyme and reference solutions werecarefully degassed immediately prior to loading of samples into thecalorimeter (reference: buffer without enzyme). Sample enzyme(approximately 1 mg/ml) and reference solutions (approximately 0.5 ml)were thermally pre-equillibrated for 20 minutes at 5° C. The DSC scanwas performed from 5° C. to 95° C. at a scan rate of approximately 90K/hr. Denaturation temperatures were determined at an accuracy ofapprox. +/−1° C.

The results as shown in Table 4 indicated that the Aspergillus nidulanslipase 1 had thermal denaturation temperatures of 63° C. in 50 mMacetate pH 5.0 buffer and 55° C. in 50 mM glycine pH 10.0 buffer.

TABLE 4 Thermostability Determination Buffer pH Td (° C.) 50 mM Acetate5.0 63 50 mM Glycine 10.0 55

Example 31 Cloning of the Aspergillus nidulans Lipase 2 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify an Aspergillus nidulans lipase 2 gene from the genomic DNAprepared in Example 24.

(SEQ ID NO: 42) Forward primer:5′-ACACAACTGGCCATGTATTTCCTTCTCTCCGTCATC-3′ (SEQ ID NO: 43) Reverseprimer: 5′-AGTCACCTCTAGTTAATTAATCAGCCTAGTGGGCAAGCAT-3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

The fragment was amplified by PCR using the Expand High Fidelity PCRSystem as described in Example 25. The reaction product was visualizedon a 0.7% agarose gel using TBE buffer and a 1.2 kb product band waspurified using a QIAquick PCR Purification Kit according to themanufacturer's instructions. The PCR product was then cloned intopCR2.1-TOPO according to manufacturer's instructions to produce pJLin170(FIG. 19). Two μl of the reaction was used to transform E. coli TOP10One Shot competent cells as described in Example 25. An E. colitransformant containing the pJLin170 plasmid was detected by restrictiondigestion and plasmid DNA was prepared using a QIAGEN BioRobot 9600.

E. coli TOP 10 One Shot cells containing pJLin170 were deposited withthe Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30754, with a deposit date of Jul. 21, 2004.

Example 32 Characterization of the Aspergillus nidulans Genomic SequenceEncoding Lipase 2

DNA sequencing of the Aspergillus nidulans lipase 2 gene from pJLin170was performed with an Applied Biosystems Model 377 XL DNA Sequencerusing dye-terminator chemistry (Giesecke et al., 1992, supra) and primerwalking strategy. Nucleotide sequence data were scrutinized for qualityand sequences were compared to each other with assistance of PHRED/PHRAPsoftware (University of Washington, Seattle, Wash.).

Gene models for the lipase gene were predicted based on homology to theThermomyces lanuginosus lipase as well as conserved sequences present atthe 5′ and 3′ ends of fungal introns. The nucleotide sequence (SEQ IDNO: 13) and deduced amino acid sequence (SEQ ID NO: 14) are shown inFIG. 20. The genomic fragment encodes a polypeptide of 308 amino acids,interrupted by 3 introns of 49, 73 and 70 bp. The % G+C content of thegene is 51.7% and the mature protein coding region (nucleotides 73 to1116 of SEQ ID NO: 13) is 51.9%. Using the SignalP software program(Nielsen et al., 1997, supra), a signal peptide of 24 residues waspredicted. The predicted mature protein contains 284 amino acids with amolecular mass of 30.5 kDa.

A comparative alignment of lipase sequences was made employing theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus nidulans lipase 2 geneshares 37% identity to the deduced amino acid sequence of a Thermomyceslanuginosus lipase gene (accession number 059952).

Example 33 Cloning of the Aspergillus nidulans Lipase 3 Gene

Two synthetic oligonucleotide primers shown below were designed based onthe predicted start and stop codons of the open reading frame to PCRamplify an Aspergillus nidulans lipase 3 gene from the genomic DNAprepared in Example 24.

(SEQ ID NO: 44) Forward primer:5′-ACACAACTGGCCATGACGGTGTCTCTTGACAGTTTATTCC-3′ (SEQ ID NO: 45) Reverseprimer: 5′-AGTCACCTCTAGTTAATTAATCACCCCCCTGACAAATCTCCCTTGG- 3′Bold letters represent coding sequence. The remaining sequence ishomologous to the insertion sites of pBM120a.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. Fifty picomoles of each of the primers above wereused in a PCR reaction containing 25 ng of Aspergillus nidulans genomicDNA. The PCR amplification reaction mixture also contained 1×PCR bufferwith 1.5 mM MgCl₂, 1 μl of a dATP, dTTP, dGTP, and dCTP mix (10 mMeach), and 0.75 μl of DNA polymerase mix (3.5 U/μl) in a final volume of501 μl. An Eppendorf Mastercycler thermocycler was used to amplify thefragment programmed for 1 cycle at 94° C. for 2 minutes; 10 cycles eachat 94° C. for 15 seconds, 62° C. for 30 seconds, and 72° C. for 3minutes; 15 cycles each at 94° C. for 15 seconds, 62° C. for 30 seconds,and 72° C. for 3 minutes plus a 5 second elongation at each successivecycle; 1 cycle at 72° C. for 7 minutes; and a 10° C. hold.

The reaction product was visualized on a 1.0% agarose gel using TBEbuffer and the 1.3 kb product band was purified using a QIAquick PCRPurification Kit according to the manufacturer's instructions.

Example 34 Construction of an Aspergillus oryzae Expression VectorExpressing Aspergillus nidulans Lipase 3 Gene

The PCR fragment containing the Aspergillus nidulans lipase 3 genedescribed in Example 33 was cloned into the pBM120a expression vectorusing an InFusion Cloning Kit. The vector was digested with restrictionendonucleases Nco I and Pac I (using conditions specified by themanufacturer). The digested vector was purified by gel electrophoresisand extracted using a QIAquick Gel Extraction Kit, and the PCR fragmentwas purified using a QIAquick PCR Purification kit. The gene fragmentand the digested vector were ligated together in a reaction resulting inthe expression plasmid pJSF8c (FIG. 21). The ligation reaction (20 μl)was composed of 1× InFusion Buffer, 1×BSA, 1 μl of Infusion enzyme(diluted 1:10), 100 ng of pBM120a digested with Nco I and Pac I, and 50ng of the Aspergillus nidulans lipase 3 gene purified PCR product. Thereaction was incubated at room temperature for 30 minutes. Two μl of thereaction was used to transform E. coli SoloPack® Gold supercompetentcells. An E. coli transformant containing the pJSF8c plasmid wasdetected by restriction digestion and plasmid DNA was prepared using aQIAGEN BioRobot 9600.

Example 35 Construction of an Aspergillus nidulans Lipase 3 Gene CloningVector

The two synthetic oligonucleotide primers described in Example 33 wereused to PCR amplify the genomic coding region of the Aspergillusnidulans lipase 3 gene from plasmid pJSF8c.

The fragment of interest was amplified by PCR using the Expand HighFidelity PCR System. Fifty picomoles of each of the primers above wereused in a PCR reaction containing 1:10 dilution of plasmid, pJSF8c, miniDNA. The PCR amplification reaction mixture also contained 1×PCR bufferwith 1.5 mM MgCl₂, 1 μl of a dATP, dTTP, dGTP, and dCTP mix (10 mMeach), and 0.5 μl DNA polymerase mix (3.5 U/μl) in a final volume of 50μl. An Eppendorf Mastercycler thermocycler was used to amplify thefragment programmed for 1 cycle at 94° C. for 2 minutes; 10 cycles eachat 94° C. for 15 seconds, 62° C. for 30 seconds, and 72° C. for 1minute, 15 seconds; 15 cycles each at 94° C. for 15 seconds, 62° C. for30 seconds, and 72° C. for 1 minute, 15 seconds plus a 5 secondelongation at each successive cycle; 1 cycle at 72° C. for 7 minutes;and a 10° C. hold.

The 1.3 kb PCR product was then cloned into pCR2.1-TOPO according tomanufacturer's instructions to produce pBM141 (FIG. 22). Two μl of thereaction was used to transform E. coli TOP10 One Shot competent cells.An E. coli transformant containing pBM141 was detected by restrictiondigestion and plasmid DNA was prepared using a QIAGEN BioRobot 9600.

E. coli TOP 10 One Shot cells containing pBM141 were deposited with theAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center, 1815 University Street, Peoria, Ill., 61604,as NRRL B-30780, with a deposit date of Oct. 12, 2004.

Example 36 Characterization of the Aspergillus nidulans Genomic SequenceEncoding Lipase 3

DNA sequencing of the Aspergillus nidulans lipase 3 gene from pJSF8c andpBM141 was performed with a Perkin-Elmer Applied Biosystems Model 377 XLAutomated DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., FosterCity, Calif.) using dye-terminator chemistry (Giesecke et al., 1992,supra) and primer walking strategy. Nucleotide sequence data werescrutinized for quality and all sequences were analyzed with assistanceof ContigExpress software (Informax, Inc., Bethesda, Md.).

Gene models for the putative lipase genes were predicted based onhomology to a Thermomyces lanuginosus lipase as well as conservedsequences present at the 5′ and 3′ ends of fungal introns. The genomiccoding sequence (SEQ ID NO: 15) and deduced amino acid sequence (SEQ IDNO: 16) are identical for pJSF8c and pBM141 and are shown in FIG. 23.The genomic fragment encodes a polypeptide of 404 amino acids,interrupted by 1 intron of 68 bp. The % G+C content of the gene is52.14% and the mature protein coding region (nucleotides 76 to 1280 ofSEQ ID NO: 15) is 52.3%. Using the SignalP software program (Nielsen etal., 1997, supra), a signal peptide of 25 residues was predicted. Thepredicted mature protein contains 380 amino acids with a molecular massof 42.4 kDa.

A comparative alignment of lipase sequences was determined using theClustal W method (Higgins, 1989, supra) using the LASERGENE™ MEGALIGN™software (DNASTAR, Inc., Madison, Wis.) with an identity table and thefollowing multiple alignment parameters: Gap penalty of 10 and gaplength penalty of 10. Pairwise alignment parameters were Ktuple=1, gappenalty=3, windows=5, and diagonals=5. The alignment showed that thededuced amino acid sequence of the Aspergillus nidulans lipase 3 geneshares 21.6% identity to the deduced amino acid sequence of aThermomyces lanuginosus lipase gene (accession number 059952).

Deposit of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli (pSMO223) NRRL B-30773Sep. 16, 2004 E. coli (pSMO224) NRRL B-30774 Sep. 16, 2004 E. colipHyGe026 NRRL B-30772 Sep. 13, 2004 E. coli pBM135g NRRL B-30779 Oct.12, 2004 E. coli pCrAm138 NRRL B-30781 Oct. 12, 2004 E. coli pJLin170NRRL B-30754 Jul. 21, 2004 E. coli pJLin171 NRRL B-30755 Jul. 21, 2004E. coli pBM141 NRRL B-30780 Oct. 12, 2004

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated polypeptide having lipase activity, selected from thegroup consisting of: (a) a polypeptide comprising an amino acid sequencehaving at least 95% identity with the mature polypeptide of SEQ ID NO: 2or SEQ ID NO: 4; (b) a polypeptide encoded by a polynucleotide whichhybridizes under at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of(i) or (ii), wherein the high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.;and (c) a polypeptide encoded by a polynucleotide comprising anucleotide sequence having at least 95% identity with the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:
 3. 2. Thepolypeptide of claim 1, which comprises the amino acid sequence of SEQID NO: 2 or SEQ ID NO: 4; or a fragment thereof having lipase activity.3. The polypeptide of claim 1, which comprises the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO:
 4. 4. The polypeptide of claim 1, whichconsists of SEQ ID NO: 2 or SEQ ID NO: 4; or a fragment thereof havinglipase activity.
 5. The polypeptide of claim 1, which consists of themature polypeptide of SEQ ID NO: 2 or SEQ ID NO:
 4. 6. The polypeptideof claim 1, which is encoded by the polynucleotide contained in plasmidpSMO223 which is contained in E. coli NRRL B-30773, or plasmid pSMO224which is contained in E. coli NRRL B-30774.
 7. The polypeptide of claim1, wherein the mature polypeptide is amino acids 25 to 396 of SEQ ID NO:2 or amino acids 25 to 283 of SEQ ID NO:
 4. 8. The polypeptide of claim1, wherein the mature polypeptide coding sequence is nucleotides 73 to1256 of SEQ ID NO: 1 or nucleotides 73 to 944 of SEQ ID NO:
 3. 9. Thepolypeptide of claim 1, comprising an amino acid sequence having atleast 95% identity with the mature polypeptide of SEQ ID NO: 2 or SEQ IDNO:
 4. 10. The polypeptide of claim 9, comprising an amino acid sequencehaving at least 97% identity with the mature polypeptide of SEQ ID NO: 2or SEQ ID NO:
 4. 11. The polypeptide of claim 1, which is encoded by apolynucleotide which hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) afull-length complementary strand of (i) or (ii), wherein the highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and 50% formamide, and washing three times each for 15minutes using 2×SSC, 0.2% SDS at 65° C.
 12. The polypeptide of claim 11,which is encoded by a polynucleotide which hybridizes under at leastvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1 orSEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or(ii), wherein the very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.13. The polypeptide of claim 1, which is encoded by a polynucleotidecomprising a nucleotide sequence having at least 95% identity with themature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:
 3. 14.The polypeptide of claim 13, which is encoded by a polynucleotidecomprising a nucleotide sequence having at least 97% identity with themature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO:
 3. 15.A detergent composition comprising the polypeptide of claim 1 and asurfactant.
 16. An isolated polynucleotide comprising a nucleotidesequence which encodes the polypeptide of claim
 1. 17. A nucleic acidconstruct comprising the polynucleotide of claim 16 operably linked toone or more control sequences that direct the production of thepolypeptide in an expression host.
 18. An isolated host cell comprisingthe nucleic acid construct of claim
 17. 19. A method for producing thepolypeptide of claim 1 comprising: (a) cultivating a host cellcomprising a nucleic acid construct comprising a nucleotide sequenceencoding the polypeptide under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.
 20. An isolatedpolynucleotide obtained by (a) hybridizing a population of DNA underhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1 orSEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or(ii), wherein the high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μg/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.;(b) isolating the hybridizing polynucleotide; and (c) determining thehybridizing polynucleotide encodes a lipase.
 21. The isolatedpolynucleotide of claim 20 obtained by (a) hybridizing a population ofDNA under very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of(i) or (ii), wherein the very high stringency conditions are defined asprehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200μ/ml sheared and denatured salmon sperm DNA, and 50% formamide, andwashing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.;(b) isolating the hybridizing polynucleotide; and (c) determining thehybridizing polynucleotide encodes a lipase.
 22. A nucleic acidconstruct comprising a gene encoding a protein operably linked to anucleotide sequence encoding a signal peptide comprising or consistingof amino acids 1 to 24 of SEQ ID NO: 2 or amino acids 1 to 24 of SEQ IDNO: 4, wherein the gene encoding the protein is foreign to thenucleotide sequence encoding the signal peptide.
 23. An isolated hostcell comprising the nucleic acid construct of claim
 22. 24. A method forproducing a protein comprising: (a) cultivating the recombinant hostcell of claim 23 under conditions conducive for production of theprotein; and (b) recovering the protein.